Investigations of microbial adaptation to singular, binary, and fully formulated quaternary ammonium compounds

ABSTRACT

The study was conducted to inform risk assessments concerning microbial exposure to quaternary ammonium biocides (QUATs) by investigating their effects on 10 microbial strains of hygiene relevance. Biocides were divided into three categories: simple aqueous solutions, biocide mixtures, and formulated biocides. Organisms were grown in the presence of biocides for 10 generations and then subsequently for another 10 generations in biocide-free media. Control organisms were passaged 20 times in biocide-free media. Strains were then assessed for biocide and antibiotic susceptibility, changes in growth dynamics, and single nucleotide polymorphisms (SNPs). Biocide mixtures demonstrated greater antimicrobial potency than singular and formulated biocides. Susceptibility changes of under twofold were observed for all biocides tested. Susceptibility decreased significantly for organisms passaged with singular biocides (1.29- to 4.35-fold) and biocide mixtures (1.4- to 1.5-fold), but not for formulated biocides (1.3- to 1.84-fold) compared to controls. Antibiotic susceptibility both increased and decreased in passaged organisms, with heightened susceptibility occurring more frequently in the singular biocide group. Changes in susceptibility and growth dynamics were similar in the passaged and unexposed controls for fitness measures of adapted bacteria; there were no significant differences between biocide groups, but significantly lower generation and doubling times in organisms exposed to singular biocides. Similar frequencies in SNPs occurred for the three biocide groups and unexposed controls. While some adaptations occurred, particularly with singular biocides, the impact on antibiotic resistance and genomic mutations was limited. These findings suggest that the use of formulated QUATs may pose a comparatively lower risk for antimicrobial resistance.

IMPORTANCE

Biocides are used globally to control microbial growth and effective assessment of the risks and benefits of their use is therefore a high priority. Much of the data used to assess risk has been based on sub-lethal exposure of bacteria to singular biocides in simple aqueous solutions. This work builds on limited prior realism-based studies to demonstrate enhanced potency in biocidal mixtures; the mitigation of resistance selection by formulations and inconsistent cross-resistance effects with both increases and decreases in susceptibility for a wide range of antibiotics. These data can be used to better inform risk assessments of biocide deployment.

INTRODUCTION

Biocides are broad-spectrum agents that are extensively used in healthcare and industry to control the growth and eliminate microorganisms. Questions have been raised about their long-term efficacy, with the possibility that prolonged exposure could select for reduced susceptibility (1, 2). Whether laboratory studies in which bacteria are exposed to biocides in simple aqueous solutions provide a reliable indication of the risks of resistance in the real world is a matter of ongoing debate (3). Ideally, risk assessments around biocide use should extend to studies that mimic the complexities of real microbial communities and likely exposure scenarios (4–7). Most studies that identify resistance risks from biocide exposure have been based on serial exposure to sub-lethal concentrations of the test compound. Recent example studies include adapted strains of Pseudomonas aeruginosa (8), Escherichia coli (9), Listeria monocytogenes (10), and various food isolates (11), while several reviews (2, 3, 12–15) refer to numerous other examples. Repeated sub-lethal exposure represents a worst-case scenario of adaptation, typically potentially resulting in susceptibility reductions to the biocide itself, and often to other antimicrobials through broad-spectrum resistance mechanisms (16). While this type of approach is relatively easy to implement, findings of reduced susceptibility and/or cross-resistance should be interpreted in the context of the methods used.

In real-life use, biocides are often deployed as mixtures of biocidal agents and as complexly formulated products with the addition of surfactants and other supplementary agents, which aim to enhance efficacy and potency. Yet compared to singular agents, the effect of formulation on the effectiveness and potential negatives of biocides as bacteriocidal agents is little studied. Furthermore, how formulation may affect biocidal cross-resistance to antibiotics and microbial growth dynamics is relatively unknown, with mixed reports (6, 7). It is hypothesized that formulated products will affect the extent and frequency of bacterial susceptibility changes, potentially mitigating decreased biocide susceptibility and off-target co-resistance to clinically relevant antibiotics.

In the current study, we use an established spiral plate method to promote the adaptation of a panel of microbial strains to quaternary ammonium biocides in pure and chemically mixed forms. Simple aqueous solutions (singular biocidal agents), mixtures of biocidal species (combinations of two or more biocides), and formulated (complex combinations of biocides and excipients) quaternary ammonium biocides were tested for susceptibility in parental and passaged microbes. For the panel of microorganisms, we chose from hygiene-relevant strains commonly used during EU product authorization testing for biocide-containing products (17). The diverse panel includes representatives of Gram-positive and Gram-negative bacteria, a Mycobacterium and a eukaryote. Strains were assessed for changes in susceptibility to the biocides to which they were exposed and for a panel of 10 antibiotics. In addition, growth curves for pre-exposure and post-exposure strains were compared for evidence of altered fitness. A “no-biocide” control passaged on biocide-free untreated agar plates served as a comparison, providing information on the effects of the passaging procedure, and hence the assessment of resistance risk.

MATERIALS AND METHODS

Microbial strains and media

The microbial strains Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC were obtained from American Type Culture Collect (ATCC). The strains Escherichia coli ATCC 10536 (DSM 682), Escherichia coli K12 NCTC 10538 (DSM 11250), Enterococcus hirae ATCC 10541 (DSM 3320), Klebsiella pneumoniae ATCC 4352 (DSM 789), and Mycobacterium smegmatis ATCC 19420 (DSM 43756) were obtained from DSMZ (Leibniz Institute, Braunschweig, Germany). Bacillus subtilis ATCC 6633 (WDCM 00003), Candida albicans ATCC 10231 (WDCM 00054), and Salmonella enterica serovar Typhimurium ATCC 14028 (WDCM 00031) were obtained as Vitroids from Merck (Merck Life Science UK Limited, Dorset, United Kingdom).

The base medium for all strains was Mueller Hinton broth and agar (MHB and MHA) (Merck). Broth additives included glucose and Tween 80 (Merck) for selected strains as discussed below. All strains were found to grow on MHA plates without supplementation after incubation at 37°C for 24 or 48 hours.

Biocides, antibiotics and antifungals

Biocides alkyldimethylbenzyl ammonium chlorides (ADBACs) of alkyl chain lengths C12 (dodecyldimethylbenzyl ammonium chloride) and C14 (tetradecyldimethylbenzyl ammonium chloride) were purchased from Merck. Didecyldimethyl ammonium chloride (DDAC) was provided by Arch UK Biocides Ltd. (50% DDAC, 20% propan-2-ol, and 30% water), from which DDAC was recrystallized and dissolved in deionised water for subsequent use. Decyltrimethyl ammonium chloride (DTAC) and ADBAC C16 (cetyldimethylbenzyl ammonium chloride) were purchased from TCI UK Ltd (Tokyo Chemical Industry UK Ltd, Oxford, United Kingdom). Alkyl (C12C14) dimethyl(ethylbenzyl)ammonium chloride (ADEBAC), biocidal mixtures, and formulated biocides were provided by Arch UK Biocides Ltd (Table 1).

TABLE 1.

Biocide compositions^a

Category	Descriptor	Composition	Biocide concentration gradient
Single active species	S1	Alkyl (C12) dimethylbenzyl ammonium chloride (ADBAC C12) (MW: 339.99 g/mol)	100 mg/mL: PA, SE 20 mg/mL: ECK682, ECK12, SA 10 mg/mL: CA 2 mg/mL: BS, KP, EH, MS
	S2	Alkyl (C14) dimethylbenzyl ammonium chloride (ADBAC C14) (MW: 368.04 g/mol)	100 mg/mL: PA, SE 20 mg/mL: BS, ECK682, ECK12, KP, SA 2 mg/mL: CA, EH, MS
	S3	Alkyl (C16) dimethylbenzyl ammonium chloride (ADBAC C16) (MW: 396.10 g/mol)	100 mg/mL: KP, PA, SE 50 mg/mL: EC682, ECK12, SA 20 mg/mL: BS, CA, EH, MS
	S4	Alkyl (C12C14) dimethyl(ethylbenzyl)ammonium chloride (ADEBAC (C12–C14)) CAS no. 85409-23-0	100 mg/mL: PA, SE 20 mg/mL: EC682, ECK12, SA 10 mg/mL: CA, KP 2 mg/mL: BS, EH, MS
	S5	40% didecyldimethylammonium chloride (DDAC) CAS no. 7173-51-5 (MW: 362.08 g/mol) 60% water	20 mg/mL: PA, SE 5 mg/mL: EC682, ECK12, SA 1 mg/mL: BS, CA, EH, KP, MS
	S6	Decyltrimethylammonium chloride (DTAC) product number D1306 (MW: 235.84 g/mol)	312 mg/mL: CA, EH, PA, SE 100 mg/mL: SA 50 mg/mL: BS, EC682, ECK12, KP, MS
Mixture of biocidal species	M1	50% alkyl(C14 50%, C16 10%, C12 40%) dimethylbenzyl ammonium chloride (ADBAC/BKC (C12–C16)) CAS no. 68424-85-1 50% water	100 mg/mL: PA, SE 20 mg/mL: CA, EC682, ECK12, KP, SA 2 mg/mL: BS, EH, MS
	M2	3.3% alkyl (C12C16) dimethylbenzyl ammonium chloride (ADBAC/BKC (C12–C16)) CAS no. 68424-85-1 3.3% alkyl (C12C14) dimethyl(ethylbenzyl) ammonium chloride (ADEBAC (C12–C14)) CAS no. 85409-23-0 3.3% didecyldimethyl ammonium chloride (DDAC C10/C10) CAS no. 7173-51-5 90.1% water and low level of alcohol solvent	20 mg/mL: PA 10 mg/mL: KP, SA, SE 5 mg/mL: EC682, ECK12 2 mg/mL: CA 1 mg/mL: BS, EH, MS
Formulation of biocidal active species	F1	6.9% didecyldimethyl ammonium chloride (DDAC) CAS no. 7173-51-5 93.1% surfactants, complexing agents, pH adjusters, solvents, water	40 mg/mL: PA, SE 10 mg/mL: CA, EC682, ECK12, SA 1 mg/mL: BS, EH, KP, MS
	F2	13% didecyldimethyl ammonium chloride (DDAC (C8-10)) [CAS no. 68424-95-3] 8.7% alkyl (C12C16) dimethylbenzyl ammonium chloride (ADBAC/BKC (C12–C16)) CAS no. 68424-85-1 78.3% surfactants, complexing agents, pH adjusters, solvents, water	40 mg/mL: PA 10 mg/mL: CA, ECK682, ECK12, EH, KP, SE 1 mg/mL: BS, SA, MS

^a Biocides are classified as single active species (S), biocidal mixtures (M), and formulated biocides (F).

Antibiotics were obtained from Oxoid (Thermo Fisher, Paisley, United Kingdom) as paper disks for disk diffusion assays. The following antibiotics were used: gentamycin (30 µg), imepenem (10 µg), ceftazidime (10 µg), piperacillin (30 µg), ticarcillin (75 µg), ciprofloxacin (5 µg), cefoxitin (30 µg), kanamycin (5 µg), tetracycline (30 µg), and cephalothin (30 µg).

Spiral plating for biocide adaptation

A WASP 2 Spiral Plater (Don Whitley, Yorkshire, United Kingdom) was used to deposit biocide solutions on the surface of MHA plates. A volume of 50 µL was deposited using the logarithmic setting, giving a 1 to 1/1,000 concentration gradient of the biocide across the radius of the plate and left to dry on the benchtop for 1 hour. Parental P0 strain was streaked across the plate horizontally and incubated aerobically at 37°C for 72 hours. A colony was then picked which grew at the highest biocide concentration (at the center of the plate) and streaked again on another plate. After 5 and 10 passages (P5 and P10, respectively) on biocide-treated plates, a sample of the inoculum was frozen at −80°C for subsequent susceptibility testing. Following P10, a further 10 passages were conducted on biocide-free MHA plates to reach PX10, at which point a growth sample was also frozen at −80°C for subsequent susceptibility testing.

MIC and MBC determination for parental and biocide-adapted strains

MIC values for P0 parental strains and biocide-exposed strains were determined by broth microdilution using MHB as per the EUCAST method (18). Broth was supplemented for C. albicans and B. subtilis with 0.5% (wt/vol) glucose (filter sterilized 0.2 µm), and for M. smegmatis 1% (wt/vol) glycerol and 2% (vol/vol) Tween 80 was added to the broth.

MBC values were determined after MIC determination, following a neutralization step. The procedure described by Knapp et al. (19) was used for neutralization, with some modifications. Briefly, using an electronic multi-channel pipette with repeat function, the entire well volume was aspirated from the MIC plate, and then 20 µL of the same fluid returned to the original well. To the 20 µL, 180 µL of sterile neutralizer (30 g L⁻¹ Tween 80 and 3 g L⁻¹ asolectin) was added and carefully pipetted to mix. The well solution was placed onto MHA-only plates and allowed to dry before being inverted and incubated at 37°C for 48 hours. MBC values were determined at 24 hours and then checked again after a further 48 hours of incubation. Susceptibility measurements for P0, P5, P10, and PX10 for a given strain and a given biocide were determined on the same day and expressed in μg mL⁻¹ of total biocidal agent.

Cross-resistance determination

Bacterial strains at P0, P5, P10, and PX10 for each biocide exposure were tested for susceptibility to each of the 10 antibiotics listed above. The disk diffusion method was used on MHA, following the EUCAST method (20). Zone diameters were measured and compared with the P0 values for the analysis of changes in susceptibility. Where breakpoints were available, these were compared to measured zone diameters for the determination of resistance.

To assess the contribution to changes in susceptibility to antibiotics from serial passaging alone, “no-biocide” control strains were also tested with antibiotics using the same method. The similarity of biocide-exposed and non-biocide-exposed strains in their antibiotic susceptibility profiles was determined by correlation analysis.

Growth curve analysis

For growth curves, each strain was grown overnight at 37°C in 10 mL of MHB (with additives for certain strains as per the MIC determination) and diluted in fresh broth to a concentration of approximately 10⁶ CFU mL⁻¹, and then 200 µL dispensed into a sterile 96-well microtiter plate. Plates were incubated for up to 48 hours in a Biotek Powerwave HT plate reader at 37°C, which recorded the 600 nm absorbance value every 30 minutes. Growth curve data were analyzed with Growth Curver (21) using R Studio which fits growth curve data to a standard form of a logistic equation common in ecology and evolution (22, 23) to ascertain population-level information including maximum possible population size (carrying capacity) and fastest possible generation time (doubling time).

Genome sequencing and variant detection of mutants

Selected strains and their parental P0 strains were sequenced using Illumina short-read sequencing. DNA extraction, library preparation, and sequencing were performed by MicrobesNG (http://www.microbesng.com, Birmingham, UK) according to their protocols. Briefly, approximately 4–6 × 10⁹ cells were concentrated and suspended in 500 µL of DNA/RNA Shield (Zymo Research, USA) in 2 mL screw cap tubes, and stored at 4°C prior to sample submission. A volume of 5–40 μL of cell suspension was lysed with 120 µL TE buffer containing lysozyme (final concentration 0.1 mg/mL) and RNase A (ITW Reagents, Spain; final concentration 0.1 mg/mL) at 37°C for 25 minutes. Protein digestion was performed using Proteinase K (VWR Chemicals, USA; final concentration 0.1 mg/mL) and SDS (Sigma-Aldrich, USA; final concentration 0.5% vol/vol) with incubation at 65°C for 5 min.

Genomic DNA was purified using an equal volume of paramagnetic beads (SPRI beads, Beckman Coulter, USA) and subsequently resuspended in EB buffer (10 mM Tris-Hcl, pH 8.0). DNA was quantified with QuantiT dsDNA HS kit (ThermoFisher Scientific, UK) using an Eppendorf AF2200 plate reader (Eppendorf, UK) and diluted to a concentration appropriate for library preparation. Library preparation was performed using the Nextera XT Library Prep Kit (Illumina, USA) according to the manufacturer’s protocol with modifications (i.e., a twofold increase in input DNA, PCR elongation increased to 45 s). DNA quantification and library preparation steps were performed using a Hamilton Microlab STAR liquid handling system (Hamilton Bonaduz AG, Switzerland). Libraries were sequenced using the Illumina NovaSeq6000 platform (Illumina, USA) using a 250 bp paired-end protocol.

Read adapter trimming was performed using Trimmomatic version 0.30 (24) with a sliding window quality cut-off of Q15. Trimmed reads were aligned to annotated reference genomes for each strain. The reference genomes for the ATCC strain S. aureus ATCC 6538 were acquired from the ATCC website. A reference genome for E. coli NCTC 10538 was acquired from NCBI GenBank (accession CP048439.1). Variants were identified using the breseq 0.38.1 pipeline with default parameter settings (25). A combined table of variants was produced using gdtools COMPARE from the breseq pipeline. Variants reported in the main text refer to changes relative to the re-sequenced parental P0 for each selected strain.

RESULTS

We exposed 10 hygiene-relevant organisms to 10 different biocide environments (six single compounds, two mixtures, and two formulations, Table 1) and a control, biocide-free environment, each in triplicate, for 10 passages, followed by a further 10 passages for all lineages in the control environment.

Changes in biocide susceptibility

MIC and MBC values of exposed and control strains are shown in supplementary Tables S1 to S4. Exposure to Singular biocides resulted in the highest mean MIC (66 µg mL⁻¹) and MBC (190 µg mL⁻¹) in comparison to mixed (MIC: 4.8 µg mL⁻¹; MBC: 7.3 µg mL⁻¹) and formulated (MIC: 18 µg mL⁻¹; MBC: 39 µg mL⁻¹) biocides. Furthermore, organisms exposed to singular biocides showed the greatest increase in MICs and MBCs after biocide exposure.

Strains with greater than a fourfold increase in MIC at P5 and/or P10 were seen in at least one organism for all singular biocides except S4 (5/6 biocides), whereas none were recorded for mixed or formulated biocides. Furthermore, the average fold change in MICs from P0 to P5 was 2.18, 1.40, and 1.41 for singular, mixed, and formulated biocides, respectively.

Changes in MICs are shown compared to the no-biocide control (NBC) (Fig. 1). For organisms exposed to singular biocides, the strains passaged against singular biocides had significantly higher MICs (P = 0.000167, F = 14.23). The organisms passaged with biocide mixtures were also found to have significantly higher MICs than control organisms (P = 0.0314, F = 4.66). Importantly, no significant differences in MIC compared to the NBC were recorded for formulated biocides (P = 0.54, F = 0.376). There were also no significant differences in MBCs in any of the biocide groups compared to the controls (singular P = 0.187 F = 1.742, mixed P = 0.302 F = 1.068, and formulated P = 0.667 F = 0.185).

Fig 1.

Organisms were passaged on concentration gradients of singular (S1–S6), mixtures (M1 and M2), and formulated biocides (F1 and F2) (Table 1), in addition to organisms passaged in the absence of biocides (controls). Susceptibility was examined at P0 (parental unpassaged strain), P5, P10, and after 10 generations passaged in the absence of biocides (PX10). Susceptibility to each biocidal agent was compared between organisms passaged with the biocides and organisms passaged in the absence of biocides. Biocide susceptibility was compared between the organisms passaged with singular, biocide mixtures, and formulated biocides to the negative controls (organisms passaged in the absence of biocides). Panels show the distribution of susceptibility to singular, biocide mixtures, and formulated biocides compared to controls. There was a significant change in the distribution of susceptibility in singular biocides (P < 0.001) and biocide mixtures (P = 0.0314) compared to controls (significantly decreased susceptibility compared to controls). The distribution of MICs was not significantly different compared to controls for formulated biocides (P = 0.54) (no significant changes in susceptibility compared to controls) (one-way ANOVA). There was significant differences when comparing biocide composition and passage to MIC (P < 0.0001, F = 27.92) and MBC (P < 0.0001, F = 11.74) (two-way ANOVA).

Changes in antibiotic susceptibility

To investigate whether repeat, sub-lethal exposure to biocides resulted in cross-resistance to antibiotics, exposed and NBC strains were considered against a panel of 10 antibiotics (Fig. 2). Further information regarding the number of times a strain recorded an increase or decrease in susceptibility to an antibiotic is shown in Tables S5 to S8.

Fig 2.

Organisms were passaged on concentration gradients of singular (S1–S6), mixtures (M1 and M2), and formulated biocides (F1 and F2) (Table 1), in addition to organisms passaged in the absence of biocides (controls). Cross-resistance was examined at P0 (parental unpassaged strain), P5, P10, and after 10 generations passaged in the absence of biocides (PX10). Distribution of antibiotics cross-resistance displayed by (A) biocide composition (P = 0.00147), (B) antibiotic (P = 0.0608), and (C) organism (P < 0.001). Values are changes in antibiotics zone size (mm) compared to the parental P0 strain.

Compared to controls, there was no significant difference in changes in antibiotic susceptibility for singular biocides (P = 0.0884), biocidal mixtures (P = 0.122), or formulated biocides (P = 0.752). The formulated group demonstrates higher incidences of decreased antibiotic susceptibility at P5 (80/180, 44.44%), P10 (81/180, 45%) (P10), and PX10 (85/180, 47.22%), compared to the singular biocide group (147/630, 23.33%) (P5), P10 (150/630, 23.81%), and PX10 (158/630, 25.08%). Mixed biocides performed similarly to formulated P5 (75/180, 41.67%), P10 (71/180, 39.44%), and PX10 (82/180, 45.56%) instances of decreased susceptibility. The NBCs had decreased antibiotic susceptibility at P5 (84/270, 31.11%), P10 (84/270, 31.11%), and PX10 (92/270, 34.07). It is important to note however that this is only the number of times decreased susceptibility was recorded and does not indicate the degree of change. There was a significant difference when comparing by biocide composition (singular vs mixed vs formulated) (P = 0.00147). There was no significant difference between antibiotics (P > 0.05) but there was by organism (P < 0.0001). S. aureus displayed the greatest and most frequent susceptibility changes (391 out of 420, 93%), with greater increases in susceptibility (306 out of 420, 73%) compared to susceptibility decreases (85 out of 420, 20%) and no changes in susceptibility (29 out of 420, 7%).

Antibiotic susceptibility data were compared to EUCAST breakpoints (v 14.0). Of the 42 comparisons available, there were some instances where disk zone sizes crossed the clinical breakpoints. Formulated biocides recorded the highest frequency of susceptibility increases for all passages (P5 n = 7, P10 n = 7, PX10 n = 10) compared to mixed (P5 n = 2, P10 n = 3, PX10 n = 3) and singular biocides (P5 n = 5, P10 n = 5, PX10 n = 5). The no-biocide control also recorded several instances of increased susceptibility (P5 n = 4, P10 n = 2, PX10 n = 3). Decreases in susceptibility were also recorded, with formulated biocides demonstrating the most instances of decreased susceptibility (P5 n = 6, P10 n = 5, and PX10 n = 8), followed by mixed (P5 n = 5, P10 n = 5, and PX10 n = 5) and singular biocides (P5 n = 2, P10 n = 3, and PX10 n = 2). The no-biocide control also recorded decreases in susceptibility (P5 n = 4, P10 n = 3, and PX10 n = 3).

S. aureus recorded the most increases in susceptibility, mostly to ciprofloxacin and tetracycline. Other notable increases in susceptibility are E. coli 682 to ceftazidime, K. pneumoniae to gentamycin, and E. hirae to ciprofloxacin.

P. aeruginosa recorded the most decreases in susceptibility, especially to imipenem and ticarcillin. S. enterica recorded multiple decreases in susceptibility to ceftazidime and piperacillin and E. coli k12 recorded numerous instances of decreased susceptibility to cefoxitin.

Growth curve analysis

To determine whether exposure to biocide resulted in a change in carrying capacity and growth dynamics, analysis was performed on growth curves of exposed strains, data are shown as a comparison to P0 (Fig. 3) (Tables S9 and S10).

Fig 3.

Organisms were passaged on concentration gradients of singular (S1–S6), mixtures (M1 and M2), and formulated biocides (F1 and F2) (Table 1), in addition to organisms passaged in the absence of biocides (controls). Growth dynamics were examined at P0 (parental unpassaged strain), P5, P10, and after 10 generations passaged in the absence of biocides (PX10). Organisms passaged in the presence of biocides were compared to parental P0 strains for change in growth dynamics. When comparing changes in carrying capacity (maximum possible population size) compared to parental P0 strains, there was a reduction in carrying capacity in the majority of biocides-exposed strains (203/270, 75%) compared to the controls (16/27, 59%) (Tables S8 and S9). There was no significant difference between biocide composition status (P = 0.904) (one-way ANOVA).

No significant difference in carrying capacity was observed between passages or biocide classification, demonstrating that these factors do not lead to a change in maximum bacterial occupancy. When Table S8 is observed, 124/162 (77%) instances of decreased carrying capacity were recorded in the singular biocide group, compared with 42/54 (78%) in the mixed biocide group and 37/54 (69%) in the formulated group. The NBC demonstrated 15/27 (56%) instances of decreased carrying capacity. C. albicans and E. hirae recorded the fewest instances of reduced carrying capacity by far (30% and 36%, respectively) with the remaining organisms displaying reduced carrying capacity in at least 76% of cases. There was no significant difference between biocide groups for K (P = 0.902, F = 0.103), but there was for t_mid (P < 0.0001, F = 18.78) and t_gen (P < 0.0001, F = 13.58) (Tables S8 and S9).

Whole-genome sequencing

Single nucleotide polymorphisms (SNPs) of S. aureus as a result of biocide exposure are shown in Fig. 4; Table S11. Mixed biocides demonstrated the largest number of SNPs at P5 on average (9.5) in comparison to singular biocides (5.8), while formulated showed the lowest (4.5). This was also the case for P10 (mixed: 10; singular: 7.7; and formulated: 6) and PX10 (mixed: 11.5; singular: 8.5; and formulated: 6.5), demonstrating that formulated biocides cause fewer SNPs for every generation. In addition, there were deletions (S1 P10, S6 P5 P10 PX10, and M1 P5 P10 PX10) and insertions (S1 P10 and M1 PX10). There was a similar frequency in SNPs in all biocides compared to the controls (Fig. 4). Repeated agrA mutations are apparent which are hypothesized to be ancestral.

Fig 4.

S. aureus passaged on concentration gradients of singular (S1–S6), mixtures (M1 and M2) and formulated biocides (F1 and F2) (Table 1), in addition to organisms passaged in the absence of biocides (controls). Genomic changes at P5, P10, and after 10 generations passaged in the absence of biocides (PX10) were compared to P0 (parental unpassaged strain). The genomes of S. aureus were sequenced using whole-genome sequencing and analyzed for SNPs, insertions, and deletions. Data indicate SNPs in S. aureus strains passaged against (A) singular, (B) biocide mixtures, and (C) formulated biocides. Each point is one of the same S. aureus strain sequenced at that passage.

DISCUSSION

The current work was partly motivated by what we perceive as shortcomings in risk assessment procedures directed toward biocide use and the potential generation of resistance and cross-resistance. Considerations include the common use of pure cultures in rich media, the use of single-agent biocides in water, the lack of biocide-free controls (or a discussion thereof), and unrealistically high culture densities.

Risk assessments that biocides could generate resistance and cross-resistance to antibiotics, including quaternary ammonium compounds, have largely focused on exposing pure cultures of bacteria to simple aqueous solutions in stepwise experiments (26, 27). However, in real-life applications, bacteria form complex communities in biofilms, and biocides are employed in complex formulations containing additives such as surfactants and sequestrants to boost efficacy. Recent studies have suggested biocide formulation can enhance antibacterial potency and mitigate the development of resistance (5, 19). In the current study, we exposed a panel of 10 microorganisms against simple aqueous, mixed, and formulated quaternary ammonium compounds, as well as control organisms passaged in the absence of biocides. The effects on biocide and antibiotic cross-resistance and associated alterations in microbial growth dynamics may reflect more accurately the way biocides interact with microorganisms in the environment and in application.

The risk of each organism developing biocide resistance was assessed by measuring fold changes in MIC and MBC. For our test biocides, we chose quaternary ammonium compounds that are commonly found in disinfectants and/or cleaning products (1, 3). Although fold changes have been displayed here, it is also important to consider the absolute concentrations. While large-fold-changes in susceptibility are often used as evidence of resistance risk, they should be seen in the context of absolute concentrations. The MIC/MBC values recorded here, between 0.0625 and 1024 µg mL⁻¹, are well below typical in-use concentrations of QUAT biocide preparations, which could be 1,500 µg mL⁻¹ and above (1, 3). Therefore, a typical cleaning product, used properly, would be expected to kill all strains identified here, especially with the addition of formulation agents that increase potency.

While antibiotic resistance has been observed following laboratory exposure to biocides, such exposure regimes are not representative of real-world exposure conditions and, as we have observed here, laboratory exposure to biocides can just as readily produce strains with increased sensitivity to antibiotics. In addition, other observers have also suggested that the link between biocide exposure and antibiotic resistance has been overstated (28).

In the current study, negative controls, compared to both singular and biocide mixtures, had increased MIC (reduced biocide susceptibility) in the biocide-exposed passaged strains. However, this was not the case for formulated biocides where there was no difference in susceptibility compared to the controls across the passages. This suggests that the formulation of quaternary ammonium compounds mitigates the reduced biocide susceptibility following sub-lethal exposure, which is associated with the unformulated quaternary ammonium compounds. The incorporation of sequestrants and surfactants into formulations appears to help mitigate the development of reduced biocide susceptibility in both extent and frequency. These findings support previous studies that report similar benefits when biocidal products are formulated (5, 19). As these additives interact with multiple bacterial targets in combination with the main active quaternary ammonium compounds, it is more of a challenge for the microbes to develop multiple physiological adaptations to reduce susceptibility (5).

The extent of reversion back toward P0 values in the absence of biocide should also be considered in the assessment of resistance risk. Overall, the extent of susceptibility changes and capacity for reversion indicates that the resistance risk for the biocides tested here is low, especially given the stringent selection pressure from repeat passaging; conditions which are unlikely to be found outside of the laboratory. Several recordings of susceptibility increases were evident following serial passaging, although these were generally less than twofold and never more than threefold. This reinforces that exposure to these quaternary ammonium biocides, even at sub-lethal concentrations, has deleterious effects that are not easily overcome.

Previous studies have at times identified a relationship between biocide exposure and antibiotic resistance (9, 10, 29, 30) and at other times not (8, 31–33), with outcomes seemingly dependent on the specific strain/biocide/antibiotic combinations used. The current study revealed a substantial number of susceptibility increases among the 10 antibiotics tested, following exposure to the biocides, and indeed even in the absence of biocide. This points to a complex, and sometimes inverse relationship between adaptation to biocides and cross-adaptation to other antimicrobials, and one in which the adaptation procedure itself plays a major part. Interestingly, it appears organisms exposed to the simple aqueous solutions exhibited more frequent increases in antibiotic susceptibility, perhaps as they were exposed to a higher QUAT active concentration without the excipients that are present in formulated biocides. However, mixed cross-resistance data are expected due to the size and complexity of a diverse microbial panel and test antibiotics, with diverse cross-resistance previously reported across different bacteria phyla (6).

Although the observation of increased sensitivity to antibiotics following biocide exposure is much less discussed, it seems as relevant as observations of decreased sensitivity. Besides the current study, Mavri et al. have reported several substantial sensitivity increases for Campylobacter species exposed to biocides (30). S. enterica strains exposed to biocides have also been reported to have increased susceptibility to antibiotics that target the cell wall (34). Uropathogenic Escherichia coli (UPEC) exposed to BAC and PHMB in some cases became more sensitive to trimethoprim-sulfamethoxazole or ciprofloxacin (35), while small colony variants of S. aureus, elicited by triclosan exposure, were found to have significantly increased antibiotic susceptibility in the majority of cases (36), although some of these changes could be attributed to culture on laboratory media due to the absence of control passages in this study. The potential for biocides to increase sensitivity to antibiotics, as these studies and the current study suggest, should form a greater part of the discussion on biocide-antibiotic cross-resistance in the future.

Reductions in carrying capacity were evident in most strains following exposure to biocides but also to a large degree for the “no-biocide” passages. This is indicative of a reduced ability or efficiency of the organisms to exploit the growth medium, following the serial passage procedure. In general, the laboratory adaptation procedure adopted here does alter the growth characteristics of the organisms, with reductions in carrying capacity and/or average growth rates typically observed. The extent to which these altered behaviors affect the competitive fitness of the adapted strains under different environmental conditions is not clear from this data but would be of interest for future study (37).

There were several SNPs in the passaged S. aureus strains which may be contributing to changes in susceptibility that warrant further investigation. Mutations in genes encoding bacitracin export ATP-binding protein, tetracycline resistance protein, and P-hydroxybenzoic acid efflux pumps may be associated with decreased susceptibility to QUATs and be involved with cross-resistance. ArgA is a quorum-sensing system in S. aureus and contributes to its pathogenicity (38). There was an SNP in this gene in several biocide-exposed and no-biocide control isolates which needs to be tested experimentally in any future work. Mutations in successive generations of passaged strains would be expected so further work is required to understand the extent they contribute to reduced QUAT susceptibility and cross-resistance in the phenotype, if at all.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.00666-24.

Supplemental material. aem.00666-24-s0001.docx.

Tables S1 to S12.

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