Listeria monocytogenes and Salmonella enterica Enteritidis Biofilms Susceptibility to Different Disinfectants and Stress-Response and Virulence Gene Expression of Surviving Cells

Abstract

Disinfection of food contact surfaces is a challenging task, aggravated by bacteria's capacity to survive and/or resist antimicrobials by means of mechanisms not yet completely understood. This work evaluated the susceptibility of Listeria monocytogenes and Salmonella enterica biofilms to four disinfectants, and analyzed how those chemical agents influenced stress-response and virulence genes expression by surviving cells. Three strains of each bacterial species mentioned were used, and their biofilms were treated with sodium hypochlorite, benzalkonium chloride, hydrogen peroxide, and triclosan using the Calgary Biofilm Device. Expression of L. monocytogenes and S. enterica stress-response genes cplC and ropS, and virulence genes prfA and avrA, respectively, was analyzed through quantitative real-time polymerase chain reaction. Results showed sodium hypochlorite to have the lowest minimum biofilm eradication concentration values (3.125 μg/ml), whereas triclosan had the worst performance since no S. enterica biofilm eradication was achieved even at the maximum concentration used (4,000 μg/ml). L. monocytogenes stress-response gene and S. enterica virulence gene were significantly upregulated in surviving cells compared with controls. In general, this work points out sodium hypochlorite as the most effective disinfectant against biofilms of both species used, and L. monocytogenes biofilms to be more susceptible to disinfection than S. enterica biofilms. Moreover, it was found that disinfection surviving biofilm cells seem to develop a stress response and/or become more virulent, which may compromise food safety and potentiate public health risk.

Introduction

Inadequate cleaning and disinfection of food-processing environments is the cause of major economic losses and represents a serious danger to public health. In fact, several studies have shown that the presence of microorganisms on food contact surfaces is one of the most common causes of food spoilage and transmission of foodborne diseases,^31,58,66 and their ability to adhere and form biofilms makes disinfection even more difficult and challenging.^19,33,81 Listeria monocytogenes and Salmonella enterica are two of the most common foodborne pathogens responsible for numerous disease outbreaks worldwide every year,^17,20,79 and numerous authors have reported that both these bacteria have the ability to adhere and form biofilms on many different surfaces.^8,30,50 Moreover, the increased difficulty in eliminating adhered and biofilm forms of these microorganisms compared with planktonic cells has also been shown in several reports.^15,22,37

The bactericidal character of most commercial products used for surfaces cleaning and disinfection is mainly based on phenolic compounds, organic acids, alcohols, chlorine, quaternary ammonium compounds (QACs), and iodophors, the efficacy of which has been reported to be higher against bacterial suspensions than against adhered cells and biofilms.^2,54,64 This fact has raised the need to reformulate the standard procedures used to test disinfectants' efficacy to include adhered cells and biofilms as targets together with planktonic cells.^23,82,83 Among the various different methods that have been used to study biofilm communities,^7,60,74 the Calgary Biofilm Device (CBD) is a high-throughput micro-titer plate-based technology for screening antimicrobial susceptibility of microbial biofilms.¹³ It was chosen to perform this work since it is a very versatile and high-throughput technique that allows minimum biofilm eradication concentration (MBEC) determination of a wide range of products and compounds such as antibiotics, biocides, metals, and disinfectants.^3,27,29

Another important issue related to surfaces disinfection is the acquisition of bacterial resistance to disinfectant agents and, further, the possible relation between chemical biocides and the emergence of resistance to antibiotics. In fact, it has been thought that some biocides and antibiotics may have similar behaviors and characteristics in the way they act and in the way bacteria develop resistance to them.^11,59,67,68 L. monocytogenes and S. enterica susceptibility and resistance to different kinds of antimicrobials has been widely studied, in both planktonic cells^1,4,40 and biofilms.^5,52,57 However, the effect of disinfection challenge on the expression of stress-response and virulence genes in these bacteria has not been so extensively studied, since only a few reports are available on this theme and all of them concern only planktonic cells.^39,78,80 Moreover, to the authors' knowledge there is no report on genetic expression analysis of L. monocytogenes or S. enterica biofilm cells after disinfection challenge. Improving the knowledge about the relation between exposure to decontaminants and genetic responses would give further information for a more correct and cautious use of decontaminants in food-processing environments. In this context, the aims of the present work were to evaluate L. monocytogenes and S. enterica biofilms susceptibility to four commonly used disinfectants, and to investigate how their action may alter surviving cells' genetic expression concerning stress-response and virulence genes.

Materials and Methods

Bacterial strains and culture conditions

To assess the behavior of different strains from different sources, this work included three L. monocytogenes (food isolate 994, clinical isolate 1562, and reference strain CECT 4031^T) and three S. enterica Enteritidis strains (food isolate 355, clinical isolate CC, and reference strain NCTC 13349). All isolates were kindly provided by Dr. Paula Teixeira (Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal). From a cryogenic stock at −70°C, strains were streaked out twice on trypticase soy agar (EMD Chemicals), and colonies were suspended in sterile saline (0.9% w/v) to match the optical density of a 0.5 McFarland standard. Suspensions were then diluted 1:30 in Mueller-Hinton II Broth cation adjusted (MH, Becton, Dickinson and Company) to a final concentration of ∼1.0 × 10⁷ colony forming unit (CFU)/ml, which subsequently served as inocula for the assays. The starting cell number was always confirmed by plating three or four replicates of serial 10-fold dilutions of a sample of the inoculum.

Calgary biofilm device

The CBD was created in 1996 by microbiologists working at the University of Calgary and consists in a batch culture technique to grow 96 equivalent biofilms at a time.^13,14 It is commercially available as the MBEC™ physiology and genetics assay (Innovotech Inc.) and consists of 96 independent pegs mounted on the inside surface of the lid of a 96-well microtiter plate. Each peg fits the corresponding well when the CBD is placed over a microtiter plate, without contacting the well surface, allowing microorganisms to grow as 96 identical biofilms. By placing the biofilms on the pegs into the wells of a microtiter plate, it is possible to assess an array of antimicrobial compounds with varying concentrations.

Biofilm formation

Single strain biofilms were grown in CBD, the pegs of which were submerged in 200 μl of inoculum placed in each well of the 96-well tissue culture plate. The device was placed on a gyratory shaker in a humidified incubator, where biofilms were left to grow at 37°C, for 24 hr at 125 rpm. After this incubation period, culture medium was discarded and biofilms on the pegs were washed for 1 min using 200 μl saline 0.9% in each well of a microtiter plate. For biofilm growth control, eight individual pegs were broken off the MBEC peg lid using sterile forceps, placed into 200 μL of recovery medium (MH + Tween 1%) and sonicated for 8 min on high with an Aquasonic (model 250HT; VWR Scientific)¹³ for biofilm disruption. Serial dilutions of the bacterial suspensions were made in 0.9% saline, plated on trypticase soy agar, and incubated for 24–48 hr at 37°C for subsequent CFUs count. Final data, given as log CFU/peg, resulted from at least three independent experiments with eight replicates each. All experimental conditions regarding biofilm formation were optimized to achieve a final biomass of about 6 log CFU/peg for all biofilms, to have countable amounts of cells even after a 3 log reduction caused by the disinfection assays.

Biofilms susceptibility tests

Disinfectants and neutralizer preparation

Four disinfectants were chosen for this study: (1) sodium hypochlorite (SH) solution, 4.99% wt/v available chlorine (Sigma-Aldrich); (2) Polycide™, a commercial product in which the active agent is benzalkonium chloride (BAC) at 6.5% w/v (Pharmax Limited); (3) hydrogen peroxide (HP) 30% w/v solution in water (Sigma-Aldrich); and (4) triclosan (Sigma-Aldrich). Working solutions were prepared fresh at maximum concentrations of 800 μg/ml for SH and BAC, 90 mg/ml for HP, and 4,000 μg/ml for triclosan. These concentrations were first selected based on previous works that also studied the action of these chemical agents on bacterial cells,^3,73 and were then adjusted if needed.

To inactivate disinfectants after biofilms challenge, it was used a universal neutralizer composed of reduced glutathione (Sigma Aldrich), L-histidine (Sigma Aldrich), and L-cysteine (Sigma Aldrich) dissolved in double-distilled water.^12,28,76 For each disinfection challenge, a fresh solution of recovery medium plus neutralizer was prepared by adding 1 volume of universal neutralizer per 40 volumes of recovery medium, with final concentrations of 2.0 g/liter reduced glutathione, 1.0 g/liter L-histidine, and 1.0 g/liter L-cysteine. The effect of the neutralizer on bacteria viability was previously tested by submerging the biofilms in recovery medium supplemented with neutralizer. Biofilms were then sonicated and a sample of the suspension was plated out to compare the number of viable biofilm cells with the controls (viable cells from biofilms not subjected to the neutralizer action). No significant reduction in bacterial numbers was observed (data not shown).

Disinfection challenge

For disinfection assays, after identical biofilms were formed as described above, biofilms were washed for 1 min with 0.9% saline to remove free cells. The disinfection challenge was then performed by submerging the biofilms in the wells of 96-well tissue culture plates containing disinfectants solutions serially diluted (twofold) in phosphate-buffered saline for 15 min, at room temperature, and without agitation. The pegs were then washed for 1 min with 0.9% saline to remove residual disinfectant solution and incubated for 1 min with the recovery medium + neutralizer (prepared as mentioned above) to inactivate the disinfectants. In the same plate, biofilms were sonicated for 8 min to promote disruption and recovery of surviving cells. Bacterial suspension dilutions and CFUs/peg counts were performed as described above.

Since for a disinfectant agent to be considered effective against adhered and biofilm cells it has to reach a 3 log units reduction,⁵¹ only the cells from biofilms that suffered such viability reduction were collected for later genetic expression analysis, as well as the corresponding biofilm cells that were not exposed to disinfection challenge (control). Moreover, for each bacterial species, only the most resistant strain to each disinfectant was selected for gene expression analysis. When different strains had the same MBEC value, the strain with the highest log CFU/peg value at the concentration immediately below MBEC was selected (data not shown). Collected cells were stored at −80°C in microtubes containing 500 μl of RNAlater^® solution (Ambion).

Genetic expression analysis

Selected genes

To obtain information about how chemical disinfection may affect the genetic expression of biofilm cells, the following virulence and stress-response-related genes were selected for each bacterial species tested. Considering L. monocytogenes, prfA was the chosen virulence gene since it is the transcriptional activator of the main virulence genes of this species,^16,21,41 with the known PrfA-regulated products, including surface proteins involved in host cell invasion and cell-to-cell spread and secreted membrane-damaging factors mediating escape from the phagocytic vacuole.^18,55 To represent the stress-response-related genes of this bacterium, it was selected the cplC gene, as it encodes a protein (CplC ATPase) that is produced under stress conditions, both inside and outside the host, and that promotes early bacterial escape from the phagosome of macrophages enhancing intracellular surviving.⁶¹

Concerning Salmonella Enteritidis, virulence expression was assessed through avrA gene, a virulence-associated gene that is located within Salmonella pathogenicity island 1 (which is necessary for the invasion of epithelial cells and induction of macrophage apoptosis), and that is involved in the induction of programmed cell death and the inflammatory response of hosts against infection.⁶ rpoS was chosen to represent the stress-response-related genes of this bacterium since it is the general stress-response regulator sigma factor required for surviving of bacteria under starvation and stress conditions.^32,34,46

Primer design

Primers used for L. monocytogenes and S. enterica stress-response and virulence genes analysis by quantitative real time-polymerase chain reaction (PCR) were designed using the software Primer 3,⁶² and are listed in Table 1. To verify the specificity of each primer pair for its corresponding target gene, PCR products were first amplified from genomic DNA (data not shown).

Table 1.

Primers Used for the Assessment of Gene Expression by Quantitative Polymerase Chain Reaction

Bacteria	Genes	Sequence (5′–3′)	Product size (bp)
Listeria monocytogenes	cplC	F: CTTGGACCTACTGGTGTTG	197
		R: TTGCCGAACTTTTTCTGTC
	prfA	F: GGTAGCCTGTTCGCTAATGA	193
		R: TAACCAATGGGATCCACAAG
	16S rRNA	F: GGAGCATGTGGTTTAATTCG	199
		R: CCAACTAAATGCTGGCAACT
Salmonella enterica Enteritidis	ropS	F: GAATCTGACGAACACGCTCA	171
		R: CCACGCAAGATGACGATATG
	avrA	F: GAGCTGCTTTGGTCCTCAAC	173
		R: AATGGAAGGCGTTGAATCTG
	16S rRNA	F: CAGAAGAAGCACCGGCTAAC	167
		R: GACTCAAGCCTGCCAGTTTC

Primers were designed using the software Primer 3.

bp, base pairs.

RNA extraction

Total RNA of each sample was extracted using the PureLink™ RNA Mini Kit (Invitrogen) according to manufacturer's recommended protocol. Potential DNA contamination was removed during RNA purification procedure by On-column PureLink™ DNase treatment (Invitrogen). RNA concentration (ng/μl) and purity (OD_260nm/OD_280nm) were assessed by spectrophotometric measurement using a NanoDrop device (NanoDrop 1000 Spectrophotometer, V3.6.0, Thermo Fisher Scientific, Inc.).

cDNA synthesis

To ensure equivalent starting amounts of RNA from control and respective treated samples to be converted to cDNA, the proper dilutions in RNase-free water were performed. cDNA of each sample was synthesized using the iScript™cDNA Synthesis Kit (BioRad). Each reaction contained 2.5 μl of iScript Reaction Mix + iScript Reverse Transcriptase and 7.5 μl of RNA template, respecting the proportions recommended by the kit manufacturer in a final reaction volume of 10 μl. Complete reaction mix was incubated in a thermocycler (MyCycler™ Thermal Cycler; BioRad) with the following reaction protocol: 5 min at 25°C, 30 min at 42°C, and 5 min at 85°C.

Quantitative real-time polymerase chain reaction

Real-time PCRs were performed on a CFX96™ Real-Time PCR Detection System Bio-Rad system (Bio-Rad Laboratories, Inc.). Each 20 μl of reaction mixture contained 2 μl of cDNA (diluted 1:20 from the cDNA synthesis reaction), 1 μl of each primer, 10 μl of 2 × SSoFast™ EvaGreen^® Supermix (Bio-Rad Laboratories, Inc.), and 6 μl of nuclease-free water. Thermal cycling conditions were as follows: 3 min initial denaturation at 95°C, followed by 40 cycles of 10 sec denaturation at 95°C, 10 sec annealing at 50°C (for L. monocytogenes samples this step was performed at 53°C, concerning primers efficiency previously determined; data not shown), and a 15 sec extension at 72°C. A melt curve was performed at the end of each run, with readings from 65°C to 95°C every 1°C for 5 sec, to confirm that only the desired product was amplified.

Gene analysis and expression

Samples for real-time PCRs were run in triplicate. Data were analyzed using the Bio-Rad CFX Manager™ version 1.6 (Bio-Rad Laboratories, Inc.) and the relative quantification method (2^−ΔΔCT),⁴⁵ which describes the change in expression of the target genes relative to the 16S rRNA reference genes from untreated control samples.^42,77 Data were analyzed by averaging the C_T values (cycle at which each sample amplification curve crosses a specific threshold) for triplicate samples. The ΔC_T values of the target genes were determined by normalizing to the endogenous control genes 16S rRNA. These samples were subsequently subtracted from the 16S rRNA genes from the untreated control samples. The ΔΔC_T was used to calculate relative expression using the formula 2^−ΔΔCT.^25,43,45 No-reverse transcriptase (no-RT) controls—RNA samples not submitted to the RT reaction—were used to check for possible DNA contamination. All no-RT controls showed ΔΔCT values above 10 cycles, confirming the quality and purity of cDNA.

Statistical analysis

Quantitative PCR data were analyzed by means of the Student's t-test, at a 95% confidence level, using the statistical program SPSS (Statistical Package for the Social Sciences).

Results

Minimum biofilm eradication concentration

Results of biofilms susceptibility to each disinfectant presented in Table 2 revealed SH to have the lowest MBEC values for all biofilms tested, ranging from 3.125 to 12.5 μg/ml. On the other hand, the lower susceptibility was found in disinfection with triclosan, since it did not eradicate any of S. enterica biofilms even at the maximum concentration used (4,000 μg/ml). An intermediate susceptibility to BAC was found comparatively with the other compounds, with notably higher MBEC values than SH but considerably lower than those registered for HP and triclosan.

Table 2.

Minimum Biofilm Eradication Concentration Values of Each Disinfectant Agent

Strains	SH (μg/ml)	BAC (μg/ml)	HP (mg/ml)	Triclosan (μg/ml)
L. monocytogenes
994	3.1	100.0	22.5	500.0
1562	6.3	50.0	45.0	500.0
CECT4031^T	3.1	6.3	11.3	250.0
S. enterica Enteritidis
355	6.3	100.0	5.6	>4,000
CC	12.5	400.0	90.0	>4,000
NCTC13349	6.3	100.0	90.0	>4,000

SH, sodium hypochlorite; BAC, benzalkonium chloride; HP, hydrogen peroxide.

Intraspecies variability was found to influence the response to each chemical agent, with some strains being predominantly more resistant to disinfection, whereas others were more susceptible. In this way, L. monocytogenes clinical isolate 1562 and S. enterica clinical isolate CC were the most resistant strains to SH and HP, and SH and BAC, respectively. On the other hand, L. monocytogenes collection strain CECT 4031^T was the most susceptible to BAC, HP and triclosan actions, whereas among S. enterica strains only food isolate 355 revealed a lower MBEC value concerning disinfection with HP (Table 2). Interspecies variability was also observed since, taking into account the average results of related strains in the same disinfection challenge, MBEC values against L. monocytogenes biofilms were inferior to those registered for S. enterica.

Stress-response and virulence gene expression

Results concerning gene expression by the most resistant L. monocytogenes and S. enterica strains to each disinfectant agent are presented in Fig. 1A, B and Fig. 1C, D, E, respectively. It was chosen to present these results graphically and per strain to enable an easier and faster observation of how each disinfectant has affected genetic expression. The first finding was that none of L. monocytogenes strains expressed the virulence gene prfA under any condition, neither before nor after disinfection (Fig. 1A-B), although its presence in genomic DNA was previously confirmed by PCR, as stated above. The same was also observed concerning expression of rpoS stress-response gene by S. enterica 355, before and after challenge with triclosan (Fig. 1C). In this way, only alterations of stress expression were registered for L. monocytogenes strains showing that, except for triclosan, all disinfectants lead to a significant increase of cplC gene expression by food isolate 994 and clinical isolate 1562. Stress expression by S. enterica strains was only notably altered in NCTC 13349 surviving cells after disinfection with HP (Fig. 1E), whereas CC biofilms treated with SH and BAC did not suffer significant alterations of rpoS gene expression (Fig. 1D). Except for SH, all disinfectants tested lead to a significant increase of virulence expression by S. enterica biofilm surviving cells, with triclosan promoting the highest increment on avrA expression, followed by HP and, finally, BAC.

FIG. 1.

Relative expression of stress-response (gray) and virulence (black) genes of Listeria monocytogenes strains (A) 994 and (B) 1562, and Salmonella enterica strains (C) 355, (D) CC, and (E) NCTC 13349. Asterisks indicate significantly different values (p < 0.05) when comparing the relative expression of control (cont) and surviving biofilm cells. BAC, benzalkonium chloride; SH, sodium hypochlorite; HP, hydrogen peroxide.

The overall results showed HP to be the disinfecting agent with more effect on stress-response and virulence gene expression, followed by BAC, whereas SH had only affected stress expression by L. monocytogenes surviving cells. Triclosan was the only disinfectant that did not interfere with cplC gene expression, but, on the other hand, it was responsible for the highest avrA upregulation in S. enterica surviving cells.

Discussion

The present work evaluated L. monocytogenes and S. enterica biofilms' susceptibility to four commonly used disinfectant agents, and analyzed stress and virulence expression by the surviving cells. Biofilms from both bacterial species were more susceptible to SH than to any other disinfectant tested. Moreover, all SH MBEC values were way below the in-use recommended concentration (200 μg/ml), ranging between 3.13 and 12.5 μg/ml. This biocidal agent is a chlorine compound used as a disinfectant, the bactericidal effect of which is based on the penetration of the chemical and its oxidative action on essential enzymes in the cell.⁴⁷ On the other hand, S. enterica biofilms were resistant to triclosan, since this was the only disinfectant tested that did not achieve biofilm eradication. This compound is a bisphenol antimicrobial agent that has a broad range of activity,⁶³ being used as a preservative, antiseptic, and disinfectant in a diverse range of products.³⁶ In this study, a concentration range of 4,000–1.95 μg/ml was used based on the fact that triclosan has been reported to be bacteriostatic at concentrations ranging between 0.025 and 100 μg/ml, and bactericidal at higher levels.^26,71,72 Although MBEC values concerning L. monocytogenes biofilms ranged between 250 and 500 μg/ml, no S. enterica biofilms eradication was achieved by triclosan even at the maximum concentration used. This performance disparity concerning the two bacterial species used might be due to the fact that Gram-negative bacteria use multiple mechanisms to develop resistance to this antimicrobial agent.^69,84 Moreover, it has been described that the main physiological change resulting from adaptation to triclosan in Salmonella is the overexpression of efflux pumps.^9,10 So, it is likely that these defensive mechanisms were taking place in S. enterica biofilm cells during disinfection and, thus, had prevented biofilm eradication.

Although not so susceptible as to SH, L. monocytogenes and S. enterica biofilms were also susceptible to BAC; most MBEC values were within the in-use recommended concentration for QACs, −200 μg/ml. BAC is a nitrogen-based surface-active QAC with a broad-spectrum antimicrobial activity. Due to their positive charge, QACs form electrostatic bonds with negatively charged sites on bacterial cell walls, destabilizing the cell wall and cytoplasmic membrane, which leads to cell lysis, leakage, and death.^49,70 Overall results obtained in this work showed a higher susceptibility of L. monocytogenes to BAC compared with S. enterica biofilms, although all S. enterica biofilms were also eradicated by this chemical agent, with only one case (CC strain) requiring a higher BAC concentration than that generally recommended.

Susceptibility tests performed with HP showed that some of its MBEC values were much higher than the 3% concentration that is generally present in disinfectants for surface wiping.³⁸ This chemical acts as a disinfectant by producing reactive oxygen species (hydroxyl radicals and superoxide anions), which attack essential cell components such as DNA, lipids, and proteins.³⁸ Although the effectiveness of peroxides against biofilms has been recognized, previous reports have also shown that HP elicited a significant microbial reduction only at concentration ranges way above the target concentrations in the commercial mixtures.^53,65,75

Having determined the MBEC of each disinfectant tested, and identified the respective L. monocytogenes and S. enterica most resistant strains, expression of stress-response and virulence genes was analyzed. The first finding was that both control and surviving biofilm cells from the L. monocytogenes strains analyzed did not express the selected virulence gene (prfA) under the conditions studied; it was also possible that the expression was below the limit of detection of the assay. Although it is not clear what controls its activity or how prfA expression is regulated, it has been reported that the regulation of PrfA and virulence gene expression is influenced by several environmental factors. One example is the temperature-dependent control of translation of the prfA messenger, which is processed only at 37°C and not at 30°C.^35,44 In the present work, disinfection challenges and collection of cells were performed at room temperature, which could be a reason why prfA expression was not detected. Moreover, intraspecies genetic expression variability is also another factor that may have caused this result, since it has been shown that genes with important functions can vary in their expression levels between strains grown under identical conditions.²⁴

On the other hand, disinfectants' actions lead to significant differences concerning the expression of stress-response genes by both bacterial species. As far as L. monocytogenes cplC gene is concerned, upregulations of almost threefold concerning SH and HP action, and twofold concerning BAC action were observed. In contrast, triclosan was the only disinfectant that did not interfere with cplC expression. As mentioned above, this gene encodes a protein that is produced under stress conditions and that promotes early bacterial escape from the phagosome of macrophages, enhancing intracellular surviving.⁶¹ So, SH, HP, and BAC actions upon L. monocytogenes biofilm cells may have triggered the same kind of stress conditions as those experienced by bacterial cells when inside a phagosome. In fact, one of the antimicrobial functions of phagocytic cells has been classified as an oxygen-dependent mechanism. Accordingly and as stated above, the mechanisms of action of SH and HP are mainly based on oxidative action, producing reactive oxygen species that attack essential cell components. In contrast, BAC acts mostly at the bacterial cells' wall and cytoplasmic membrane, destabilizing them and leading to death through cell lysis. A similar threat is presented to L. monocytogenes inside a phagosome, where lysozyme acts directly on the bacterial cell wall proteoglycans present especially in the exposed cell wall of Gram-positive bacteria.⁴⁸

Regarding the genetic analysis of S. enterica biofilms, the expression of the stress-response gene rpoS was only significantly increased after disinfection with HP. Besides being the general stress-response regulator sigma factor, rpoS has been reported to play an important role in biofilm formation,⁵⁶ which infers that its upregulation after treatment with HP may be a response to the damage caused by the free radicals produced by this chemical agent in the biofilm matrix.⁸³ Among S. enterica biofilms that were genetically analyzed, those formed by strain 355 were the only ones that did not express the rpoS gene. As stated above concerning prfA gene expression, intraspecies gene expression variability is a likely reason of this occurrence.

Finally, the analysis of avrA gene expression by S. enterica biofilms showed that disinfection with triclosan, HP, and BAC leads to significant upregulations of about six-, five-, and twofold, respectively, compared with controls. However, SH was the only disinfectant that did not promote notable modifications on the expression of this gene. The substantial upregulation of this gene observed after treatment with triclosan is in agreement with a previous study that reported S. typhimurium biofilms response to this antimicrobial to include changes of gene expression.⁷³ In this way, our results not only corroborate these previous findings but also highlight that such bacterial response is not exclusively triggered by triclosan, since the same kind of genetic alteration was observed regarding S. enterica biofilms disinfection with HP and BAC.

Conclusions

The overall results of this work showed L. monocytogenes and S. enterica biofilms to be more susceptible to SH than to any other disinfectant tested, whereas all S. enterica biofilms were resistant to triclosan within the concentration range used. Save this case, all disinfection challenges were influenced by intra- and interspecies variability, as denoted by the different MBEC values observed after challenged with each disinfectant. Moreover, the overall results showed that the most resistant strains to each disinfection challenge had undergone genetic adjustments in terms of stress-response and/or virulence, depending on the bacterial species and strain. Consequently, the main finding of this work is the interesting and worrying fact that, even at concentrations that lead to significant reduction in biofilm biomass, disinfectants may induce virulence of the surviving cells and, thus, increase their infectious potential in case of contact with a host. Nevertheless, these studies are yet preliminary, since a higher number of strains need to be tested to address the high variability observed and, thus, reach more definite conclusions. Likewise, a wider range of target genes and disinfectants need to be studied to confirm the conclusions presented here, and to clarify which specific factors inherent to disinfection can be triggering the genetic changes of biofilm surviving cells.

Footnotes

Acknowledgments

Authors gratefully acknowledge the helpful contribution of Joana Bento and Ângela França on the quantitative PCR assays. The authors acknowledge the financial support from Portuguese Foundation for Science and Technology through grants SFRH/BD/28887/2006 and SFRH/BPD/26803/2006, respectively.

Disclosure Statement

The authors have no conflicts of interest to declare.

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