Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence

Abstract

Objectives

To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics.

Methods

Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry.

Results

Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB–TolC efflux system after PFD exposure.

Conclusions

Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB–TolC efflux system, but that single exposure was insufficient to select for MAR strains.

MAR, efflux, proteomics, biocide resistance

Introduction

In two recent studies, we demonstrated that growth of Salmonella enterica and Escherichia coli with subinhibitory concentrations of farm disinfectants led to a small but statistically significant increase in isolation of multiply antibiotic-resistant (MAR) strains, which were detected when subsequently exposed to cyclohexane or antibiotics.^1,2 In E. coli and S. enterica serovar Typhimurium (hereafter referred to as Salmonella Typhimurium), MAR is associated with reduced susceptibility to antibiotics such as β-lactams, chloramphenicol, fluoroquinolones and tetracyclines, increased tolerance to organic solvents and decreased susceptibility to disinfectants such as pine oil.^3–5 In E. coli, overexpression of the efflux pump acrAB and regulators marA and soxS can all lead to MAR.² Although the mechanisms of MAR in S. enterica are not so clearly defined as for E. coli, the same resistance phenotype is seen in isolates from farms and farm animals.⁴

In recent years, there have been increasing concerns that disinfectant exposure may help drive selection of antibiotic-resistant bacteria.^6,7 Disinfectants are often a mixture of active compounds and as a result usually have multiple, intracellular targets, therefore it has been proposed that disinfectant resistance is unlikely to arise from a single mutational event as can occur with antibiotic resistance.⁸ Consequently, for high-level disinfectant resistance to arise as a result of target site mutations repeated selection events and alteration of a number of target proteins may be necessary. Previous studies have demonstrated that overexpression of multidrug xenobiotic efflux transporters (e.g. AcrAB–TolC) with broad substrate specificity can confer decreased resistance to antibiotics, dyes, disinfectants and detergents.⁹ The selection of MAR is a concern as such strains may be able to act as a stepping stone to high-level resistance in E. coli and S. enterica.^1,4–5

In our previous studies, however,^1,2 there were essentially two selection stages, the first being growth in subinhibitory concentrations of disinfectants and the second being plating the strains on either media with 4× MIC of antibiotics or media overlaid with cyclohexane. We were not able to isolate strains with reduced susceptibility to the farm disinfectants themselves, nor were we able to isolate MAR mutants after exposure to farm disinfectants, without the additional exposure to either antibiotics or cyclohexane. In a farm environment it is possible that bacteria will be exposed to both antibiotics and disinfectants and that over time multiple exposures may occur, so we were interested in whether limited exposure to farm disinfectants alone could select antibiotic-resistant strains.

The aim of this study, therefore, was to select strains of Salmonella Typhimurium from agar containing four different commonly used farm disinfectants and to compare the phenotype of mutant and parent strains with respect to efflux, antibiotic and disinfectant susceptibility, growth in the presence of subinhibitory concentrations of disinfectants, protein expression and the ability to survive and persist in chicks. We attempted to select mutants with increased disinfectant tolerance from a relevant panel of strains including ciprofloxacin-resistant and -susceptible isolates, Salmonella Typhimurium phage type DT104 isolates as well as from defined mutants in which efflux pump genes have been inactivated.

Materials and methods

Strains, media and chemicals

The panel of strains used to select for disinfectant tolerant mutants comprised a mixture of wild-type and laboratory mutants as well as animal isolates. As ciprofloxacin resistance is a problem in Salmonella Typhimurium,¹⁰ ciprofloxacin-resistant mutants and isolates were included, also representative DT104 isolates were included in the panel. In order to analyse the contribution of active efflux to intrinsic and high-level disinfectant tolerance, mutants lacking either acrB or tolC were also included. E. coli I113 (NCTC 10418) and Salmonella Typhimurium SL1344 were used as susceptible control strains for all susceptibility testing experiments. E. coli AG100 and AG102 were used as controls for cyclohexane tolerance assays.²⁵ All strains used in this study are listed in Table 1 and were routinely cultured on Luria–Bertani (LB) agar and in LB broth unless stated otherwise. All chemicals were obtained from Sigma-Aldrich (Poole, Dorset, UK) except for ciprofloxacin, which was kindly donated by Bayer (Newbury, Berkshire, UK). The farm disinfectants used were a tar oil phenol (PFD) which was a blend of high boiling point tar acids and organic acids, an oxidizing compound based disinfectant (OXC), an aldehyde based disinfectant (ABD) and a dairy sterilizing disinfectant (DSD) which comprised a quaternary ammonium biocide, non-ionic surfactant and excipients.

Table 1

Strains used in this study to select mutants and control strains

Designation	Description	Reference
Parent strains for mutant selection
L354	SL1344; originally isolated from a calf with diarrhoea	22
L108	SL1344; tolC::aph	23
L643	SL1344; acrB::aph	15
L696	SL1344; laboratory-selected, ciprofloxacin-resistant mutant (GyrA Asp-87→Gly)	11
L699	SL1344; laboratory-selected, cyclohexane-resistant mutant	24
L357	DT104; representative isolate, antibiotic-susceptible	24
L358	DT104; representative pentaresistant isolate, ciprofloxacin-resistant (GyrA Ser-83→Phe)	24
L378	ciprofloxacin-resistant field isolate (GyrA Ser-83→Phe)	this study
Control strains
AG100	E. coli K12	25
AG102	AG100 (cyclohexane-tolerant, ΔmarR)	25
I113	E. coli K12 (NCTC 10418)	14

Selection of mutants after single exposure to disinfectants

Mutants were selected as previously described.¹¹ Parent strains were grown overnight in antibiotic-free broth, concentrated by centrifugation and re-suspended in sterile broth to give a range of inocula (10⁶–10¹⁰ cfu/mL). Agar plates containing disinfectants at 2× the MIC (v/v; Table 2) were inoculated with 100 µL (10⁵–10⁹ cfu) of each cell suspension and incubated at 37°C in air for up to 7 days. Ten colonies with the typical size and morphology of the original strain were chosen randomly from each selecting plate and subcultured onto disinfectant-free media and retained for further study of the mechanism of resistance.

Table 2

Susceptibility of mutants to antibiotics and disinfectants and times for a 5 log reduction in viable numbers

								Disinfectant susceptibility
		Antibiotic susceptibility MIC (mg/L)						time (min) for 5 log kill				MIC (%, v/v)
Strain	Selective agent	CIP	CHL	TET	EtBr	AF	KAN	OXC (0.6%, v/v)	ABD (0.025%, v/v)	DSD (0.2%, v/v)	PFD (0.075%, v/v)	OXC	ABD	DSD	PFD
L354	NA (parent)	0.015	4	1	1024	128	2	8.2 ± 0.8				0.4	0.025	0.1	0.2
OXC-1	OXC	0.015	8	0.5	1024	128	2	11.5 ± 1				0.8
L358	NA (parent)	0.5	>256	128	1024	256	2		12.1 ± 0.2			0.8	0.025	0.8	0.2
ABD-1	ABD	2	>256	256	1024	256	2		14.2 ± 0.7				0.025
L378	NA (parent)	4	>256	256	2048	256	2			38 ± 3.2		0.4	0.025	1.6	0.2
DSD-1	DSD	4	>256	128	1024	256	2			34 ± 2.1				>3.2
L108	NA (parent)	<0.015	1	0.5	16	8	>32			15 ± 0.8		0.4	0.006	<0.003	0.025
DSD-2	DSD	<0.015	1	0.5	16	8	>32			25 ± 1.2				0.12
L357	NA (parent)	0.015	2	1	512	128	2				14.5 ± 0.5	0.4	0.025	0.1	0.2
PFD-1	PFD	0.015	4	1	1024	128	8				6.2 ± 0.3				0.2

CIP, ciprofloxacin; CHL, chloramphenicol; TET, tetracycline; EtBr, ethidium bromide; AF, acriflavine; KAN, kanamycin; OXC, oxidizing compound; ABD, aldehyde based farm disinfectant; DSD, dairy sanitizer disinfectant; PFD, phenolic farm disinfectant.

Values in bold indicate changes in MIC of antibiotics or disinfectants for mutants compared with their respective parent strains or changes in the time required to provoke a 5 log reduction in viable numbers; ± values indicate standard deviation from the mean.

Calculation of disinfectant exposure times required for a 5 log kill

Disinfectant activity was quantified by determining the ability of a disinfectant to reduce the viable numbers of a suspension of bacteria. The decimal reduction time (D value) is the time required for a specified concentration of disinfectant to lead to a 1 log (90%) reduction in viable numbers.¹² In this study, the ability of each disinfectant used to kill mutants with decreased susceptibility was determined using an assay based on the European suspension test which requires a 5 log (99.999%) kill (EN1040). Initial experiments defined the concentrations of each disinfectant required to give a 5 log reduction in viable numbers of bacteria within 30 min. These conditions were then used to compare the time required for a 5 log kill for mutant and parent strains. For each strain, an overnight culture in LB broth was diluted to ∼5 × 10⁸ cfu/mL, 1 mL of this suspension was then added to 8 mL of test disinfectant solution and 1 mL of sterile water. At 5 min time intervals, 0.5 mL aliquots were removed and added to universal tubes containing 0.5 mL of sterile water and 4.5 mL of neutralizing buffer.¹² Each suspension created in this way was serially diluted in sterile water and aliquots plated onto LB agar plates, which were incubated overnight at 37°C before colony numbers were recorded. Viable counts were performed in parallel from the original cultures used in order to allow enumeration of the numbers of cells killed after disinfectant treatment. Viable count data were also used to correct for differences in sizes of the original inocula within each experiment in order to allow comparison of data from parent and mutant strains. Data were analysed using Excel (Microsoft, USA) to calculate means and standard deviations for each strain. Differences between strains were analysed for statistical significance using the Student's t-test.

MICs and cyclohexane resistance

Organic solvent (cyclohexane and hexane) tolerance was determined as previously described.⁴ MIC testing of antibiotics and farm disinfectants used the BSAC agar doubling dilution method.¹³

Cell envelope analysis

The expression of major outer membrane proteins and lipopolysaccharide was investigated as described previously¹⁴ and patterns compared between parent and mutant strains. All strains were also visualized microscopically in order to detect any filamentation or other gross morphological changes.

Sequencing of quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE

The QRDRs of gyrA, gyrB, parC and parE from ABD-1 with cross-resistance to ciprofloxacin were amplified and sequenced as previously described.¹⁵

In vitro analysis of growth kinetics

The ability of parent and disinfectant tolerant strains to grow in the presence of concentrations of disinfectants at MIC and sub-MIC concentrations was monitored in LB broth using a FLUOstar OPTIMA (BMG LABTECH, UK). Sterile LB broth (100 µL) was dispensed into sterile microtitre trays and inoculated with overnight culture of each strain to give a final inoculum of 4% v/v. Readings were taken every 10 min of the optical density of each well (scanned at 600 nm) in the microtitre trays over the 24 h time period and results recorded automatically. Each strain was analysed in triplicate wells on at least three separate occasions to give nine data sets for analysis. Additionally, each strain was challenged with either 0.5× or the MIC of the selective biocide for the parent after 2 h of growth (mid-logarithmic growth phase) in order to determine whether the inhibitory ability of each biocide was reduced in mutants, respective to parent strains. Data were analysed using Excel (Microsoft, USA) to calculate means and standard deviations for each strain. Differences between strains were analysed for statistical significance using the Student's t-test.

Accumulation of norfloxacin

As it is a good substrate of the AcrAB–TolC system and easily detected¹⁶ the accumulation of norfloxacin by each strain in the presence and absence of CCCP (carbonyl cyanide-m-chlorophenylhydrazone, which dissipates the proton motive force and hence acts as an inhibitor of active efflux) was directly measured as previously described.¹⁶

Effect of disinfectant exposure on the proteome of SL1344

Protein expression was assessed in order to determine bacterial stress response after disinfectant treatment. Proteomes were prepared in triplicate from SL1344 with and without exposure to PFD (0.04%, v/v) and OXC (0.15%, w/v) disinfectants for 90 min. Protein extracts were prepared and analysed by 2D-LC-MSⁿ as described previously.^17,18 The relative abundance of the proteins was compared using the spectrum count method¹⁹ following published guidelines²⁰ and denotes the number of peptide counts (‘hits’) detected for each protein. Protein expression was given as a ratio of the spectrum count. Expression analysis was limited to only those proteins common to all three replicates from control or disinfectant-treated cultures. Proteomes were compared using Microsoft Access and Excel. The statistical significance of percentage changes in protein expression was determined using the two-tailed Student's t-test.

Survival of mutant strains in chicks

Chick studies were performed in a similar manner to previous studies.²¹ In brief, at three weeks of age, each group of birds was infected by intra-gastric gavage with 10⁶ cfu per bird with either strain DSD-1 or PFD-1 or OXC-1 (Table 2). For each of these groups of birds, there was a control group of birds infected with the respective parent strain (Table 2). Cloacal swabs were taken from each bird twice weekly for up to 27 days after inoculation using pre-weighed swabs, which were weighed after swabbing so that results gave cfu/g of caecal contents. Swabs were plated onto Rambach agar (Merck, Darmstadt, Germany), which was incubated overnight at 37°C. As chicks were kept together in groups, for statistical analysis, individual birds were regarded as independent units that did not interact with any other birds in the same group as far as the infection is concerned. With this assumption, the non-parametric Mann–Whitney was used to compare the mean log counts of the parent and disinfectant tolerant strains for each day post-infection. All animal studies were conducted under the jurisdiction of the Animals Scientific Procedures Act (1986) and were reviewed by the local ethics review committee.

Results

Mutants with increased disinfectant tolerance were selected

Disinfectant tolerant mutants were selected from a range of strains (Tables 1 and 2) at a frequency between 1 × 10⁻⁹ and 1 × 10⁻¹⁰. It was only possible to select disinfectant tolerant strains from agar containing 2× MIC of disinfectants, not from agar with 4× or higher multiples of disinfectant MICs. In general, multiple mutants with an identical phenotype were selected from each parent, when this was the case, data for one representative mutant has been displayed (Table 2).

Disinfectant, antibiotic and organic solvent susceptibility

L108 (SL1344, tolC::aph) and L643 (SL1344, acrB::aph) were both hyper-susceptible to the phenolic (PFD), quaternary ammonium (DSD) and aldehyde (ABD) based disinfectants with ≥8-fold increases in susceptibility to these agents with respect to SL1344, but were not more susceptible to the oxidative disinfectant (OXC). There was little variation in the susceptibility of the other strains to disinfectants; the ABD was significantly (~10-fold) more active than the other agents tested (Table 2).

Using the European suspension test (EN1040), three of the five mutants required statistically significantly longer times to generate a 5 log reduction in viability than their respective parents (Table 2). OXC-1 required longer exposure to OXC for a 5 log kill (Figure 1) and the MIC of OXC was one-dilution higher in this mutant than its parent (Table 2). Mutants isolated after exposure to ABD displayed a 4-fold increase in MIC of ciprofloxacin as well as requiring longer exposure to ABD for a 5 log kill (Table 2). Mutants obtained after exposure to DSD were more resistant to exposure to DSD as seen by MIC (DSD-1) or increased time required to obtain a 5 log kill (DSD-2). Interestingly, DSD-2 was obtained from L108 (tolC::aph) after exposure to DSD and was significantly more resistant than its parent to DSD but not to the other agents tested where it retained the hyper-susceptibility seen in L108 after inactivation of tolC (Table 2). Mutant PFD-1 obtained after exposure to PFD showed an increased tolerance to chloramphenicol, ethidium bromide and kanamycin but was not more tolerant to disinfectants; in fact this mutant actually required a shorter PFD contact time to provoke a 5 log reduction in viable cell numbers, which is an unexplained observation.

Figure 1

Survival of SL1344 (white bars) and OXC-1 (black bars) after exposure to OXC (0.6%). *P > 0.05 relative to parent strain at the same time point.

No increased organic solvent tolerance was detected for any of the disinfectant-selected mutants compared with their respective parent strains (data not shown).

Growth kinetics and accumulation assays

Accumulation of norfloxacin was unchanged for all mutants apart from ABD-1 (derived from L358 after exposure to ABD), which accumulated significantly less norfloxacin than their parent (Figure 2). The addition of CCCP increased norfloxacin accumulation in ABD-1 mutant indicating that the reduced accumulation seen in these strains is likely to be a consequence of active efflux.

Figure 2

Norfloxacin accumulation. White bars, −CCCP; black bars, +CCCP. *P > 0.05 relative to parent strain at the same time point.

None of the disinfectant-selected mutants was compromised in their ability to grow in LB broth relative to their parents in the absence of disinfectant. The disinfectant-selected mutants were more resistant to the addition of disinfectants to the media than their respective parent strains, including those strains for which the MIC of the selective disinfectant had remained unchanged when compared with the parent. DSD-selected mutant DSD-2 was able to grow significantly (P > 0.01) better upon both the addition of 0.1% and 0.2% DSD to the media (Figure 3). ABD-selected mutant ABD-1 and OXC-selected mutant OXC-1 both grew significantly (P > 0.01) faster than their parents when challenged with 0.5× or the MIC of ABD or OXC (data not shown). No significant differences were observed between PFD-selected mutant PFD-1 parent L357 in disinfectant-free broth or when exposed to 0.5× the MIC of PFD. However, PFD-1 did grow significantly (P > 0.05) better when challenged with the MIC of PFD (data not shown).

Figure 3

Growth of L108 and DSD-2 with and without exposure to DSD. Each point is the average of nine values; error bars are omitted for clarity. The arrow indicates the time at which DSD was added to each well. Grey symbols, L108; open symbols, DSD-2; triangles, no disinfectant; squares, 0.1% DSD; circles, 0.2% DSD.

Mechanism of increased ciprofloxacin resistance in ABD mutants

The frequency of mutant selection of ABD-1 (and other mutants selected at the same time from the same parent with an identical ciprofloxacin resistance phenotype) was consistent with a one- step point mutation. The parent of ABD-1, L358, carries a Ser-83→Phe substitution within GyrA; despite the increased resistance to ciprofloxacin in ABD-1, no additional changes were found in the QRDRs of any of the topoisomerase genes investigated. Investigation of the LPS and OMP profiles of all three ABD mutants indicated no changes compared with L358 demonstrating that loss of porins was not responsible for increased ciprofloxacin resistance in these strains. The accumulation data (Figure 2) suggests that increased efflux activity alone is responsible for this increased ciprofloxacin resistance.

Different disinfectants provoke different changes in the proteome of SL1344

As the MIC data (Table 2) suggested that PFD, DSD and ABD are subject to active efflux but OXC is not, the effect of the PFD (0.04%, v/v) and the OXC (0.15%, w/v) disinfectants on the proteome of SL1344 was determined. Treatment with both disinfectants had no significant effect on the total number of proteins detected. For the PFD experiment, a mean number of 360 proteins were detected in both the treatment and control (no disinfectant) and 424 proteins were detected in the OXC experiment (Table 3). The expression of 12 and 32 proteins were significantly (P > 0.05) altered following treatment with the PFD and OXC disinfectants, respectively (Tables 3 and 4). The pattern of protein expression was very different after exposure to the two disinfectants. The PFD disinfectant significantly (P < 0.05) increased expression of proteins consistent with efflux being required for disinfectant tolerance including the AcrAB/TolC efflux pump, EmrA another multidrug efflux pump protein and others associated with detoxification of hydroperoxides (AhpC and AhpE) and pyruvate dehydrogenase (AceE and AceF) (Table 4). The OXC disinfectant increased expression of a range of proteins including RpsC, a 30S ribosomal subunit; HtrA, a periplasmic serine protease; and FhuA, a transporter for ferrichrome (Table 5). Both disinfectants increased expression of PqiB the paraquat-inducible protein.

Table 3

Summary of protein changes after disinfectant exposure

	Number of proteins
Exposure	control (mean ± SD)	treated (mean ± SD)	control only	treatment only	common to treatment and control	increased expression (P < 0.05)	decreased expression (P < 0.05)
0.04% v/v PFD	437 (696 ± 129)	429 (682 ± 69)	79	70	360	12	23
0.15% w/v OXC	596 (927 ± 91)	511 (861 ± 45)	173	88	424	32	67

Table 4

Proteins with statistically significantly altered expression after treatment with PFD

Group	Description	Protein	Ratio
Efflux
	RND family, multidrug efflux pump	AcrB	2.8**
	multidrug efflux pump	EmrA	2.7**
	membrane fusion protein of AcrAB–TolC	AcrA	1.9**
	outer membrane efflux channel	TolC	1.8***
	OmpF assembly	AsmA	0.5*
ATP synthesis
	membrane-bound ATP synthase, F1 sector, β-subunit	AtpD	0.9*
	membrane-bound ATP synthase, F0 sector, subunit b	AtpF	0.8*
Chemotaxis
	methyl-accepting chemotaxis protein I	TsR	0.4**
	methyl-accepting transmembrane citrate/phenol chemoreceptor	TcP	0.3***
	methyl-accepting chemotaxis protein II	CheM	0.2***
Hydrogenase/dehydrogenase
	pyruvate dehydrogenase, dihydrolipoyltransacetylase component	AceF	2.2**
	pyruvate dehydrogenase, decarboxylase component	AceE	2.0***
	putative hydrogenase, membrane component	OmpA	1.3*
	2-oxoglutarate dehydrogenase	SucA	0.5*
	plasma membrane proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase	PutA	0.3**
Miscellaneous
	sigma D (sigma 70) factor of RNA polymerase	RpoD	2.5*
	alkyl hydroperoxide reductase	AhpC	2.2**
	fructose-bisphosphatase	Fbp	1.7*
	50S ribosomal subunit protein L6	RplF	1.6*
	pyruvate kinase I	PykF	1.4*
	transcriptional activator of ntrl gene	OsmE	0.8*
	DNA-binding protein HU-α	HupA	0.7*
	succinyl-coa synthetase, β-subunit	SucC	0.7***
	part of modulator for protease specific for ftsh phage lambda cii repressor	HflC	0.6**
	glycoprotein/polysaccharide metabolism	YbaY	0.6*
	ATP-dependent protease, Hsp 100	ClpB	0.6*
	phosphoenolpyruvate carboxykinase	PckA	0.5*
	periplasmic l-asparaginase II	AnsB	0.5*
	glycerophosphodiester phosphodiesterase	GlpQ	0.5*
	mannose-specific enzyme IID	ManZ	0.4*
	putative outer membrane lipoprotein	STM1607	0.4*
	glutathione oxidoreductase	Gor	0.4*
	stringent starvation protein A, transcriptional regulator	SspA	0.4**
	aspartate ammonia-lyase (aspartase)	AspA	0.4*
	scaffolding protein for murein-synthesizing holoenzyme	MipA	0.4*

Ratio indicates protein expression after disinfectant treatment divided by expression in untreated control.

*P < 0.05.

**P < 0.01.

***P < 0.001.

Table 5

Ten most overexpressed and repressed proteins after treatment with OXC

Description	Protein	Ratio
30S ribosomal subunit protein S3	RpsC	8.0***
Uptake of enterochelin; tonB-dependent uptake of B colicins	ExbB	6.5***
Membrane-bound lytic murein transglycosylase A	MltA	3.8***
Subunit of cysteine synthase A and O-acetylserine sulfhydrolase A	CysK	3.6***
ABC superfamily (atp&memb), cytochrome-related transporter	CydC	3.5**
Periplasmic serine protease Do, heat shock protein	HtrA	3.5**
Glucose dehydrogenase	Gcd	3.3*
Outer membrane protein receptor/transporter for ferrichrome, colicin M, and phages T1, T5, and phi80	FhuA	3.3***
Putative outer membrane lipoprotein	YiaD	2.6**
Putative helicase	RhlB	2.4*
Phosphoenolpyruvate synthase	Pps	0.2*
Glycine cleavage complex protein P, glycine decarboxylase	GcvP	0.2*
Putative methyl-accepting chemotaxis protein	STM3138	0.1**
Methyl-accepting chemotaxis protein III, ribose and galactose sensor receptor	Trg	0.1**
l-Lactate dehydrogenase	LldD	0.1*
Periplasmic l-asparaginase II	AnsB	0.1**
Phosphoenolpyruvate carboxykinase	PckA	0.1***
Fumarate reductase, anaerobic, flavoprotein subunit	FrdA	0.1*
Flagellar biosynthesis; flagellin, filament structural protein	FliC	0.1***
Putative methyl-accepting chemotaxis protein	STM3216	0.1***

Ratio indicates protein expression after disinfectant treatment divided by expression in untreated control.

*P < 0.05.

**P < 0.01.

***P < 0.001.

Disinfectant-selected mutants are fit in the chick model

There were no statistically significant differences between the ability of the parent and disinfectant mutant strains in their ability to colonize and persist in chicks (data not shown). All groups of birds were still shedding ~10⁶ cfu of Salmonella/g of caecal contents at the end of the experiments, 27 days after infection indicating that mutants colonized and persisted as well as parent strains.

Discussion

In this study, mutants of Salmonella Typhimurium with a stable increased tolerance to disinfectants were selected from a range of strains at a frequency which suggests one mutational event and that a single target of each disinfectant has been altered in each mutant. As the disinfectants used all have multiple modes of antibacterial action, it is likely that selection of mutants highly resistant to disinfectants will require multiple exposures to disinfectant and successive selection events.

The MIC data indicated that an intact AcrAB–TolC system contributes to intrinsic resistance to PFD, DSD and ABD but not OXC. The proteomic data support this conclusion with increased levels of AcrAB–TolC and EmrA detected after exposure to PFD but not after exposure to OXC, which would appear to be insensitive to active efflux. The selection of mutant DSD-2 (selected from L109, tolC::aph) with a level of tolerance to DSD similar to most strains, although it lacks a functional tolC gene, suggests that another efflux system may compensate for the loss of TolC in this mutant and that secondary transporters, other than AcrAB–TolC, may also be able to transport DSD. These data suggest that different disinfectants may differ with respect to their potential to select for mutants which overexpress efflux pumps.

None of the disinfectant-selected mutants was MAR, indicating that short, single exposure to these agents was insufficient to select for such mutants. In previous studies, disinfectant exposure followed by antibiotic exposure did lead to selection of MAR.^1,2 However, the pattern of proteins whose expression was increased by exposure to the PFD was consistent with those previously associated with MAR,^1–5 including increased expression of AcrAB–TolC. These data suggest that there is a link between disinfectant exposure and the major effectors of MAR. These first-step mutants with a low level of disinfectant resistance could act as a stepping stone to MAR strains.

The pattern of increased protein expression after treatment with OXC and PFD was distinct reflecting the different active constituents of each disinfectant. Any overlap was limited to increased expression of PqiB the paraquat-inducible protein which is a member of the soxRS regulon and therefore may have roles in both response to oxidative stress and regulation of acrAB. Although PFD induced expression of several efflux proteins, OXC increased expression of different proteins including NfnB (dihydropteridine reductase/oxygen-insensitive NAD(P)H nitroreductase) and AhpF (alkyl hydroperoxide reductase, F52a subunit; detoxification of hydroperoxides), which have been associated with resistance to hydrogen peroxide, this is likely to reflect a stress response provoked by the oxidizing compounds contained by the OXC.

The increase in the MIC of ciprofloxacin seen for mutant ABD-1, after exposure to ABD, is likely to be a result of decreased accumulation of ciprofloxacin in these mutants, as no topoisomerase substitutions in addition to the Ser-83→Phe substitution present in the parent or alterations in porin expression were found. The accumulation of norfloxacin was reduced in these strains compared with their parent and the susceptibility of these strains to CCCP suggests that active efflux is responsible for the reduced norfloxacin accumulation observed. No decreased susceptibility to ciprofloxacin was detected in any of the other mutants selected from other strains after exposure to ABD (data not shown) suggesting that the GyrA substitution in L358 may predispose selection of higher level ciprofloxacin resistance as seen in ABD-1. The potential for disinfectants to drive resistance to ciprofloxacin is a real concern⁶ and may provide a selective pressure for the selection or maintenance of ciprofloxacin-resistant strains in the farm environment in the absence of ciprofloxacin itself.

None of the disinfectant-selected mutants was compromised in their ability to grow in vitro and was better able to tolerate challenge with disinfectants. The fitness of disinfectant tolerant strains in chicks was not compromised. A key consideration for assessing the risk that any mutants may present is their ability to survive in different food production environments. These may include the general farm environment, and associated specific niches, and the animals themselves where different selective pressures may apply. The present observation that the fitness of the disinfectant tolerant mutants was not compromised in chickens is a cause for concern as survival in birds would enable both persistence on the farm and transit through the food chain.

Funding

This work was supported by the UK Department for Environment, Food and Rural Affairs, grant reference numbers OD2010 and OD2011 to M. J. W., N. G. C. and L. J. V. P.

A. C. M. is supported by grant GA678 from the BSAC to M. A. W. and L. J. V. P.

Transparency declarations

None to declare.