Use of sodium lauroyl sarcosinate (sarkosyl) in viable real-time PCR for enumeration of Escherichia coli

Use of sodium lauroyl sarcosinate (sarkosyl) in viable real-time PCR for enumeration of Escherichia coli

Journal of Microbiological Methods 98 (2014) 89–93 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: ww...

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Journal of Microbiological Methods 98 (2014) 89–93

Contents lists available at ScienceDirect

Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

Use of sodium lauroyl sarcosinate (sarkosyl) in viable real-time PCR for enumeration of Escherichia coli Hui Wang, Colin O. Gill, Xianqin Yang ⁎ Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000C & E Trail, Lacombe, Alberta, T4L 1 W1, Canada

a r t i c l e

i n f o

Article history: Received 13 December 2013 Received in revised form 7 January 2014 Accepted 7 January 2014 Available online 17 January 2014 Keywords: Escherichia coli Verotoxigenic E. coli Detergent Sarkosyl Sodium deoxycholate Propidium monoazide

a b s t r a c t The cell membranes of inactivated Escherichia coli are not always permeable to propidium monoazide (PMA). This limits the use of PMA real-time PCR (PMA-qPCR) for quantification of DNA from only viable cells for enumeration of E. coli. The aim of this study was to develop PMA-qPCR procedures for E. coli with improved selectivity for viable cells. E. coli inactivated by incubation at 52 °C were treated with 12 detergents before PMA treatment, and DNA was quantified by real-time PCR. Treatment with each of the 12 detergents and PMA increased the cycle threshold (Ct) values for heat inactivated E. coli suspensions. The greatest increase, of 10.68 Ct was obtained with sarkosyl. Treatment with sodium deoxycholate (NaDC) increased the Ct value by 8.99 Ct. Treatment with sarkosyl or NaDC of 16 heat treated 5-strain cocktails of verotoxigenic E. coli (VTEC) increased the mean Ct values by 8.15 or 6.82 Ct, respectively. Those mean values were significantly (p b 0.05) different. When used to enumerate viable E. coli in suspensions treated with lactic acid or in mixtures of viable E. coli and E. coli inactivated by peroxyacetic acid, the slopes relating the Ct values from sarkosyl treated samples to the numbers of viable E. coli were 2.24 and 2.47, respectively, with regression coefficient values ≥0.85. The findings show that sarkosyl was more effective than NaDC for dissipation of PMA-barrier properties of membranes of inactivated E. coli cells. Viable E. coli in mixtures of viable E. coli and E. coli inactivated by heat, lactic acid or peroxyacetic acid could be reliably enumerated by sarkosyl PMA-qPCR. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Ethidium monoazide (EMA) and propidium monoazide (PMA) are dyes that can intercalate with DNA and, upon exposure to light, form covalent bonds with the bases (Nocker and Camper, 2006; Nocker et al., 2006). Any crosslinked DNA that can be extracted is inactivated for PCR. Consequently, treatment of bacterial suspensions with a monoazide dye before extraction of DNA has been used to exclude extraneous DNA and DNA in dead cells from quantification by real-time PCR (qPCR). The selective reaction of the monoazide dyes with DNA in dead but not in live cells depends upon the dyes being unable to cross the membranes of living cells to react with the DNA within them (Nocker et al., 2007a). Some viable cells can be permeable to EMA (Kobayashi et al., 2009). Therefore, use of PMA for selective inactivation for PCR of DNA in dead cells is preferred (Lee and Levin, 2009a). However, treatments that are lethal for bacteria do not necessarily result in changes to cell membranes that render them permeable to PMA. Such treatments include exposure to relatively mild but ultimately lethal heating or oxidizing conditions or relatively low concentration of oxidizing agents that are ultimately lethal (Løvdal et al., 2011; Nocker et al., 2007b; Yang et al., 2011). In North America, bacteria on beef are commonly exposed to conditions of those sorts during routine ⁎ Corresponding author. Tel.: +1 403 782 8119; fax: +1 403 782 6120. E-mail address: [email protected] (X. Yang). 0167-7012/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2014.01.004

decontamination of beef carcasses and cuts (Gill, 2009). Rapid determination of the efficacies of such treatments for assessment of the microbiological safety of beef would be desirable. This might be done by qPCR for DNA in only those cells that survived a decontaminating treatment. However, such a viable qPCR is not possible by treatment of microbiological samples with PMA alone, because the PMA treatment will not affect DNA in all dead cells. It has been shown that the ability of membranes of dead cells to prevent entry of PMA can be overcome by treating the cells with sodium deoxycholate (Lee and Levin, 2009b; Yang et al., 2011). Escherichia coli, generally tolerates concentrations of deoxycholate that are lethal for other bacteria (Thanassi et al., 1997), although the bile tolerance of E. coli is probably variable between strains (Bidlack and Silverman, 2004). Thus, for E. coli, deoxycholate is the obvious membrane disrupting agent to use with viable PCR. The disruption of cell membranes by deoxycholate is due to its action as a detergent (Begley et al., 2005). The mechanisms that render E. coli tolerant of deoxycholate confer tolerance of other detergents also. It is then possible that detergents other than deoxycholate might give better selective disruption of intact membranes of dead cells or have a more consistent effect on the membranes of viable cells. As information on the use of other detergents for this purpose is lacking, the matter was investigated. The study was carried out using E. coli because of its status as an indicator organism for enteric pathogens and the importance of verotoxigenic E. coli (VTEC) as enteric pathogens associated with beef. Detergents that included 9 bile salts and an anionic,

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a non-ionic and a zwitterionic detergent, were tested for their efficacies for rendering E. coli inactivated by incubation at 52 °C permeable to PMA. Subsequently, the efficacies of a selected detergent for viable qPCR with cocktails of VTEC incubated at 52 °C were determined. The suitability of the selected detergent in conjunction with PMA-qPCR for enumeration of viable E. coli cells in suspensions exposed to 4% lactic acid or ≤90 ppm of the oxidizing agent peroxyacetic acid (PAA) was investigated. The lactic acid treated suspensions of E. coli were resuscitated before they were treated with detergent and PMA because lactic acid may permeabilize the cell membranes of viable E. coli (Alakomi et al., 2000; Shi et al., 2011). 2. Materials and methods 2.1. Preparation of cell suspensions A wild type strain of E. coli that had been isolated from meat (Jones et al., 2002) was grown to the stationary phase in 10 ml of half strength brain heart infusion (BHI; Difco, Becton Dickinson, Sparks, MD, USA). The cultures were incubated for 16 h at 37 °C, with shaking at 80 rpm. Serial tenfold dilutions of each culture to 10−7 were prepared in 0.1% (w/v) peptone water. Duplicate 0.1 ml portions of the 10−6 and 10−7 dilutions were spread on plates of tryptone soya agar (TSA; Oxoid, Nepean, ON, Canada). The plates were incubated at 35 °C for 24 h, and colonies on plates bearing 25 to 250 colonies were counted. The numbers of viable E. coli were determined from those counts. Appropriate dilutions of cultures in peptone water were prepared for each of the treatments. 2.2. Preparation of detergent solutions Sodium cholate (NaC), sodium chenodeoxycholate (NaCDC), sodium deoxycholate (NaDC), sodium glycocholate (NaGC), sodium glycochenodeoxycholate (NaGCDC), sodium glycodeoxycholate (NaGDC), sodium taurocholate (NaTC), sodium taurochenodeoxycholate (NaTCDC), sodium taurodeoxycholate (NaTDC), Triton X-100 and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) were each dissolved in sterile, 0.1% (w/v) buffered peptone water to obtain 5% (w/v) solutions. A solution of sodium lauroyl sarcosinate (sarkosyl) was prepared similarly, but at a concentration of 2.5% (w/v). All detergents with the exception of Triton X-100 (MP Biomedicals, Solon, OH, USA) were obtained from Sigma-Aldrich (Sigma-Aldrich, Oakville, ON, Canada). 2.3. Determination of efficacies of detergent treatments for rendering mild heat inactivated E. coli permeable to PMA One-milliliter portions of a suspension of the wild type strain of E. coli (106 cfu ml−1) were dispensed to 2 sets of 26 2-ml Eppendorf tubes. One set of tubes was kept in ice water. The other set was incubated in a water bath operated at 52 ± 0.1 °C for 4.5 h. This treatment was expected to result in a N6 log reduction in E. coli numbers (Yang et al., 2011). Immediately after removal from the water bath, the tubes were cooled in ice water, then centrifuged at 10,000 ×g for 3 min. Each pellet was resuspended in 240 μl of 0.1% peptone water. Duplicate suspensions were each mixed with 60 μl of one of the detergent solutions or 0.1% peptone water. All the tubes were incubated in a 37 °C water bath for 30 min. The suspensions were then treated with PMA, as described previously (Yang et al., 2011). After completion of the PMA treatment, cells were pelleted by centrifugation. DNA extracted from pellets was quantified by real-time PCR using primers for the uidA gene, as described previously (Yang et al., 2011). 2.4. Effects of sarkosyl treatment on VTEC isolates Suspensions of each of 37 VTEC strains that had been isolated from the feces or the oral cavities of beef cattle (Aslam et al., 2010) were prepared as were suspensions of the wild type E. coli. Sixteen 15-ml test

tubes that each contained 10 ml of half strength BHI were each inoculated with 5 μl of each of 5 VTEC suspensions selected at random from the 37 available suspensions. Thus, each isolate was included in at least two of the 16 5-strain VTEC cocktails. After incubation overnight at 35 °C, ten-fold serial dilutions of each cocktail of cultures to 10− 3 in 0.1% peptone water were prepared. A 1 ml portion of each 10− 3 dilution was dispensed to each of 8 2-ml Eppendorf tubes. Four tubes were kept in ice water and the other 4 were heated at 52 ± 0.1 °C for 4.5 h, as before. Then, ten-fold serial dilutions of each suspension that was held on ice were prepared, as before. Duplicate 0.1 ml portions of suitable dilutions of the suspensions that were not heated and duplicate 0.1 ml portions of the undiluted heated suspensions were spread on plates of TSA, for determination of the number of viable VTEC. Portions of each heated or not heated suspension were treated with PMA, sarkosyl and PMA or NaDC and PMA, as described before. DNA was extracted and quantified by real-time PCR using primers for the uidA gene, as was DNA from the wild type E. coli. 2.5. Quantification of viable E. coli in E. coli suspensions treated with lactic acid Suspensions (108 cfu ml−1) of the wild type strain of E. coli overnight cultures were prepared, as before. A solution of 4.4% lactic acid (SigmaAldrich) in sterile, distilled water was prepared. Portions of the solution were adjusted to pH 2.4, 2.8, 3.2, 3.6 or 4.0, by addition of 3 N NaOH. A 1-ml portion of the suspension was mixed with 9 ml of the lactic acid solution of each pH. The acidified cell preparations of pH 2.4, 2.8, 3.2, 3.6 or 4.0 were incubated at room temperature for 5, 20, 40,150 or 360 min, respectively. At appropriate intervals, a 1-ml portion of each preparation was withdrawn and mixed with 9 ml of 0.1% peptone water supplemented with 0.04 M dipotassium phosphate. The pH values of the neutralized preparations were 6.9 ± 0.1. Four 1-ml portions of each neutralized preparation were centrifuged at 10,000 ×g for 3 min. Each pellet was resuspended in 1 ml of half strength BHI and incubated at 37 °C for 2 h to resuscitate the cells. After resuscitation, the cells in each tube were pelleted by centrifugation, as before. The pellets in two of the tubes were suspended in peptone water, and ten-fold dilutions in peptone water to 10−3 were prepared. Duplicate plates of TSA were spread with 0.1 ml portions of the undiluted suspension and each dilution. Plates were incubated and colonies were counted as before, for enumeration of E. coli that survived the acid treatment. Pellets in each of the other two tubes were suspended in 240 μl of 0.1% peptone water, and then were treated with sarkosyl and PMA, as before. When treatments with sarkosyl and PMA had been completed the mixtures were centrifuged, as before. DNA from each pellet was quantified by real-time PCR using primers for the uidA gene, as before. 2.6. Quantification of viable E. coli in mixtures of viable E. coli and E. coli inactivated by PAA Solutions of PAA (Sigma-Aldrich) at 20, 50 and 100 ppm in distilled water were sterilized by filtration through a 0.22 μm syringe membrane-filter (Nalgene, Thermo, MA, USA). A 9-ml portion of each solution was mixed with a suspension (108 cfu ml−1) of an overnight culture of the wild type E. coli. The mixtures were incubated at room temperature for 30 min. A 3-ml portion of each preparation was withdrawn and mixed with 27 ml of neutralizing buffer (Difco) supplemented with 0.04 M thioglycolate. A 1-ml portion of each neutralized preparation was used to prepare ten-fold serial dilutions to 10− 3, in peptone water. Duplicate plates of TSA were spread with 0.1 ml portions of the undiluted neutralized preparation and each dilution, for enumeration of E. coli. Four 1-ml portions of each neutralized preparation were centrifuged, as before, and each pellet was resuspended in peptone water and treated with PMA. Upon completion of PMA treatment, the mixtures were centrifuged to obtain pellets, as before. Extracted DNA

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was quantified by real-time PCR using primers for the uidA gene, as before. Portions of each of the neutralized E. coli preparations of suspensions that had been mixed with 50 or 100 ppm PAA were supplemented with a suspension of viable E. coli to obtain mixtures in which the proportions of viable cells were 0.001%, 0.01%, 0.1%, 1.0%, 10% or 50% of the total (1.0 × 106 cells ml− 1). Cells in the viable suspension, each inactivated suspension and each mixed suspension were pelleted and resuspended in 240 μl of 0.1% peptone water. The suspensions were treated with sarkosyl and PMA. Upon completion of treatments, cells were pelleted as before. Extracted DNA was quantified by real-time PCR, using primers for the uidA gene, as before. The numbers of viable cells in each mixture was determined by spreading 0.1 ml portions of each serially diluted or undiluted suspension on plates of TSA, as before. 2.7. Data analysis E. coli counts were transformed to log values. Mean cycle threshold (Ct) values from real-time PCR were plotted against means for log E. coli counts. Regression slopes and correlation coefficient values (R2) for those plots were obtained using Microsoft Excel 2010 (Microsoft. Redmond, WA, USA). A Ryan-Joiner test in Minitab (version 16; Minitab Inc., Sate College, PA, USA) for normal distribution was applied to each set of Ct values for VTEC cocktails. Differences in mean Ct values were separated using a paired t-test in Minitab. 3. Results Treatments of suspensions of viable E. coli with most detergents before treatment with PMA did not result in substantial increases of Ct values (Fig. 1). Treatments with CHAPS before treatment with PMA, however, increased the Ct value by 3.80 Ct. For the heat inactivated suspensions, treatments with NaDC before treatment with PMA increased the Ct value by 8.99 Ct, which was the highest among the increases resulting from treatment with bile salts; and treatment with sarkosyl before treatment with PMA increased the Ct value by 10.68 Ct. The increases in Ct values by treatments with NaCDC, NaGDC or NaGCDC and PMA were between 5.07 and 6.11 Ct. Treatments with the other 7 detergents did not cause substantial increases in Ct values. The extents to which the treatments with the three unconjugated bile salts before treatment with PMA increased Ct values obtained with suspensions of E. coli killed by the mild heat treatment were in the order NaDC N NaCDC N NaC. Treatments with glyco- or tauro-conjugates of bile salts before treatment with PMA resulted in Ct values for suspensions of

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dead E. coli that were similar to or less than the Ct values obtained by treatment with the corresponding unconjugated bile salt before treatment with PMA. After VTEC cocktails were incubated at 52 °C for 4.5 h, no VTEC were recovered from 7 of the cocktails. That is, the reductions in cell numbers were N5 log units. The reductions in cell numbers of VTEC in the other 9 cocktails were between 2.7 and 4.2 log units. For all 16 cocktails, the differences in Ct values resulting from treatments with sarkosyl or NaDC before treatment with PMA were normally distributed (p N 0.05), irrespective of whether or not the suspensions were incubated at 52 °C. For suspensions not incubated at 52 °C, the differences resulting from sarkosyl or NaDC treatment before treatment with PMA were mostly ≤−0.6 and ≤0.4 Ct, respectively (Table 1). The means and standard deviations for the difference were −0.6 ± 0.25 and 0.6 ± 0.56 for treatment with sarkosyl or NaDC, respectively, before treatment with PMA. For suspensions incubated at 52 °C, treatment with either detergent before treatment with PMA increased the Ct values significantly (p b 0.05) as compared with treatment with PMA only (Table 1). The mean changes in Ct values as results of treatments with sarkosyl or NaDC before treatment with PMA were 8.15 and 6.82 Ct, respectively. Those mean values were significantly (p b 0.05) different. For resuscitated cells in lactic acid treated suspensions of the wild type E. coli, the regression coefficient (R2) for the plot relating the log cell numbers to the Ct values for DNA obtained from them when they were treated with sarkosyl before treatment with PMA was 0.93 (Fig. 2). The slope of the plot was 2.24 Ct log cfu−1. No E. coli were recovered from suspensions that were treated with any of the PAA solutions. Ct values for DNA extraction from suspensions of E. coli that were or were not treated with ≤45 ppm PAA, and were or were not treated with PMA were similar. However, when the suspensions were treated with 90 ppm PAA, the difference in Ct values for DNA from cells that were or were not treated with PMA was about 4 Ct. When cells were inactivated by incubation with PAA at concentrations of 45 or 90 ppm PAA, treatment with sarkosyl before treatment with PMA increased Ct values for DNA obtained from the cells by 6.0 and 3.7 Ct to 28.8 and 29.2 Ct, respectively. When mixtures containing b0.1% viable E. coli were treated with sarkosyl before treatment with PMA, the Ct values were between 28.8 and 31.6 Ct, irrespective of the numbers of viable cells. However, when the numbers of viable cells were ≥0.1% of the total, the R2 value for the plot of Ct values against log cell numbers was 0.85 and the slope was 2.47 Ct log cfu−1 (Fig. 3).

Table 1 Changes in cycle threshold (ΔCt) values resulting from treatments with sarkosyl (SK) or sodium deoxycholate (NaDC) of suspensions (106 cfu ml−1) of verotoxigenic Escherichia coli cocktails (C) that were not heat treated (NT) or were heated (HT) at 52 °C for 270 min. Suspensions

Fig. 1. Effects of treatment with detergents before treated with propidium monoazide (PMA) on cycle threshold (Ct) values for DNA preparations from suspensions of live Escherichia coli (□) or E. coli inactivated by incubation at 52 °C (■). The differences in Ct values were derived by subtracting Ct values for suspensions that were not treated with detergent and/or PMA from those that were treated with both.

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16

Reductions (log cfu)

5–6

2.7–4.2

NT (ΔCt)

HT (ΔCt)

SK

NaDC

SK

NaDC

−0.61 −0.86 −1.04 −0.53 −0.53 −0.34 −0.19 −0.88 −0.90 −0.46 −0.50 −0.53 −0.38 −0.93 −0.47 −0.47

−0.01 1.70 0.15 0.93 0.76 0.93 1.08 0.25 1.32 1.45 0.08 0.05 0.37 0.26 0.18 0.13

11.04 9.68 9.15 9.13 9.41 8.23 10.41 9.20 7.95 7.28 6.85 7.33 6.19 6.37 6.32 5.92

8.97 9.21 6.86 6.21 6.77 5.12 7.56 7.25 6.81 7.52 5.58 5.38 8.65 5.33 4.50 7.46

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Fig. 2. Plot of the numbers of Escherichia coli recovered from suspensions treated with 4% lactic acid at pH 2.4 (◊), 2.8 (○), 3.2 (□), 3.6 (■) or 4.0 (●) against the Ct values for DNA obtained from the acidified suspensions that were incubated in half strength brain heart infusion at 37 °C for 2 h, when treated with sarkosyl and propidium monoazide before extraction of DNA. Bars show the standard deviation and for symbols without bars, the standard deviation falls within the symbol.

4. Discussion The tolerance of bile salts and other detergents by E. coli is due to the barrier properties of the outer membrane and the system for active efflux of any such substances that do enter the cell (Begley et al., 2005; Thanassi et al., 1997). The effects of detergents on the membranes of dead E. coli are dependent on only the emulsifying properties of the detergents, that is, their ability to bind with lipids and proteins of the cell membrane and so disrupt the membrane structure. Membrane disruption by a detergent is therefore related to the detergent's hydrophobicity. The pKa values of the unconjugated bile acids deoxycholic, chenocholic and cholic acids are 6.58, 6.4 and 6.2, respectively (Hofmann and Roda, 1984; Ko et al., 1994). Consequently, large fractions of these substances are uncharged at pH 7.0. Conjugation of the bile acids with glycine or taurine lowers the pKa values by 2.4 and 5.0, respectively. Therefore, large fractions of the conjugated bile salts are ionized at pH 7.0 and are less hydrophobic than their respective unconjugated forms. The finding that NaDC, NaCDC and NaC were more efficacious than their respective conjugated forms for rendering heat inactivated E. coli permeable to PMA was then likely a result of the differences in the pKa values of the unconjugated and conjugated acids. The hydrophobicity of the unionized bile acids decreases in the order NaDC N NaCDC N NaC, in accordance with the numbers, positions and orientations of hydroxyl groups in the steroid skeletons (Begley et al., 2005). The trihydroxy bile salt NaC is less hydrophobic than the dihydroxy bile salts NaDC and NaCDC. Thus, the finding that NaC was the

Fig. 3. Plot of the cycle threshold (Ct) values for mixtures of viable Escherichia coli and E. coli inactivated by 45 ppm (○) or 90 ppm (●) peroxyacetic acid against the numbers of viable E. coli, when the mixtures were treated with sarkosyl and PMA before DNA extraction. Bars show the standard deviation and for symbols without bars, the standard deviation falls within the symbol.

least, and NaDC was the most effective of the three bile salts for rendering heat inactivated E. coli permeable to PMA could be expected. Sarkosyl is an anionic detergent with an aliphatic hydrophobic moiety and a pKa of 3.6. It was then considered that it would likely be more efficacious for membrane disruption of dead cells than NaDC, which is a rather mild detergent. The reported maximum concentration of sarkosyl that does not have adverse effect on viability of E. coli is 0.5% (Keyhani and Keyhani, 2010). In this study, 0.5% sarkosyl was used and was confirmed to have minimal effect on E. coli viability. The treatment of heat inactivated E. coli with 0.5% sarkosyl increased the Ct value by 10.68 Ct, which is an increase comparable with that previously reported for difference between Ct values for DNA from viable cells and Ct values for DNA from cells inactivated by incubation at 90 °C and treated with PMA (Yang et al., 2011). Incubation of E. coli at 90 °C apparently disrupts cell membranes uniformly to allow unimpeded access of PMA to intracellular DNA. Thus, the sarkosyl treatment was found to be similarly effective to heat at 90 °C for largely eliminating the PMA-barrier properties of the cell membranes of E. coli inactivated by mild heat treatments. Populations of bacteria from cultures that have not been exposed to antimicrobial treatments will include some injured and moribund cells (Francois et al., 2005). These cells could be rendered permeable to PMA by exposure to a detergent. Thus, some Ct value increase as a result of treating viable cultures with detergent and PMA, as was found for viable VTEC cultures treated with NaDC and PMA could be expected. However, the unexpected decreased Ct values found when viable VTEC cultures were treated with sarkosyl before treatment with PMA suggest that sarkosyl may protect injured but viable cells from entry of PMA. This could perhaps occur as a result of electrostatic interactions between the negatively charged sarkosyl and positively charged PMA that prevented PMA from entering injured but viable cells. Further investigation of this matter is required, but the findings suggest that loss of DNA for PCR from injured but viable cells will be less when sarkosyl rather than NaDC is used to treat cells before they are treated with PMA. Moreover, the greater variation in Ct values for samples treated with NaDC than for samples treated with sarkosyl suggests that the effect on E. coli of sarkosyl is less strain dependent than the effect of NaDC. The findings of this study on the effects of sarkosyl and NaDC treatments on various strains of E. coli therefore indicate that sarkosyl is superior to NaDC with respect to consistency and efficacy when used to differentiate viable and dead E. coli for PMA-qPCR. Substantial fractions of viable cells of E. coli that had been treated with lactic or acetic acid were found to be permeable to a DNAintercalating dye (Shi et al., 2011). However, the membrane barrier properties were restored when the treated cells were incubated in a complex medium. It then appears that E. coli exposed to organic acid must be incubated under conditions that allow repair of damaged cell membranes before exposure to PMA treatment if the numbers of viable cells of E. coli are to be properly estimated by PMA-qPCR. In this study, the acidified E. coli suspensions were resuscitated in half strength BHI before treatment with sarkosyl and then with PMA. The slope relating the Ct value to log cfu is comparable to the corresponding plot previously obtained for E. coli surviving heat treatments (Yang et al., 2011). Thus, the numbers of E. coli that survived lactic acid treatments were apparently accurately quantified by sarkosyl PMA-qPCR. Treatment of E. coli with the oxidizing agent PAA at 90 ppm resulted in damage to the membranes of a sizable fraction of cells sufficient to allow PMA permeation. However, E. coli killed by incubation with PAA at ≤45 ppm were evidently largely impermeable to PMA. Thus, as has been reported for Salmonella treated with NaClO (Nocker et al., 2007b) and heat treated E. coli (Shi et al., 2011; Yang et al., 2011), relatively mild but lethal treatments with PAA can leave dead E. coli with membranes that remain impermeable to PMA. At packing plants in North America, beef carcasses, cuts and conveyor belts are often sprayed with 200 ppm PAA, which is the maximum concentration allowed by regulation for application to meat (Gill, 2009; USDA, 2013; Yang et al.,

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2012). The actual concentration of PAA to which bacteria on meat or equipment surfaces are exposed is less than 200 ppm and not uniform, because of dilution by surface water and the rapid reaction of PAA with all forms of organic matter at surface (Briñez et al., 2006). Thus, a population of E. coli with heterogeneous membrane permeability would be expected on carcasses or cuts that are sprayed with PAA. The maximum reduction in the number of E. coli on beef that can be obtained by spraying carcasses or trimmings with 200 ppm PAA is ≤ 1 log (Ellebracht et al., 2005; Gill, 2009; King et al., 2005). This is well within the range for reliable enumeration by the sarkosyl PMA-qPCR method of viable E. coli in mixtures of viable E. coli and E. coli that are inactivated by PAA. Therefore, the findings of the study show that rapid determination of the effects of treatments for decontamination of beef by heat, or solutions of organic acids or oxidizing agents using sarkosyl PMA-qPCR should be practicable. Possibly the method could have wider application for rapid determination of the effects of relatively mild processing condition or treatments used for control of pathogens in other foods. Acknowledgements We thank Ms. M. Rajagopal for assistance with real-time PCR assays. Funding for this study was provided by the Alberta Livestock and Meat Agency and Agriculture and Agri-Food Canada. References Alakomi, H.-L., Skyttä, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K., Helander, I.M., 2000. Lactic acid permeabilizes Gram-negative bacteria by disrupting the outer membrane. Appl. Environ. Microbiol. 66, 2001–2005. Aslam, M., Stanford, K., Mcallister, T.A., 2010. Characterization of antimicrobial resistance and seasonal prevalence of Escherichia coli O157:H7 recovered from commercial feedlots in Alberta, Canada. Lett. Appl. Microbiol. 50, 320–326. Begley, M., Gahan, C.G., Hill, C., 2005. The interaction between bacteria and bile. FEMS Microbiol. Rev. 29, 625–651. Bidlack, J.E., Silverman, P.M., 2004. An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts. J. Bacteriol. 186, 5202–5209. Briñez, W.J., Roig-Sagués, A.X., Hernández Herrero, M.M., López-Pedemonte, T., Guamis, B., 2006. Bactericidal efficacy of peracetic acid in combination with hydrogen peroxide against pathogenic and non pathogenic strains of Staphylococcus spp., Listeria spp. and Escherichia coli. Food Control 17, 516–521. Ellebracht, J.W., King, D.A., Castillo, A., Lucia, L.M., Acuff, G.R., Harris, K.B., Savell, J.W., 2005. Evaluation of peroxyacetic acid as a potential pre-grinding treatment for control of Escherichia coli O157:H7 and Salmonella Typhimurium on beef trimmings. Meat Sci. 70, 197–203. Francois, K., Devlieghere, F., Standaert, A.R., Geeraerd, A.H., Cools, I., Van Impe, J.F., Debevere, J., 2005. Environmental factors influencing the relationship between optical density and cell count for Listeria monocytogenes. J. Appl. Microbiol. 99, 1503–1515.

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Gill, C.O., 2009. Effects on the microbiological condition of product of decontaminating treatments routinely applied to carcasses at beef packing plants. J. Food Prot. 72, 1790–1801. Hofmann, A.F., Roda, A., 1984. Physicochemical properties of bile acids and their relationship to biological properties: an overview of the problem. J. Lipid Res. 25, 1477–1489. Jones, T., Gill, C.O., Mcmullen, L., 2002. The behaviour of log phase Escherichia coli at temperatures below the minimum for sustained growth. Food Microbiol. 19, 83–90. Keyhani, J., Keyhani, E., 2010. Increased resistance to detergent in Enterococcus faecalis. In: Mendez-Vilas, A. (Ed.), Proceedings of the International Conference on Antimicrobial Research. World Scientific, Valladolid, Spain, pp. 46–50. King, D.A., Lucia, L.M., Castillo, A., Acuff, G.R., Harris, K.B., Savell, J.W., 2005. Evaluation of peroxyacetic acid as a post-chilling intervention for control of Escherichia coli O157: H7 and Salmonella Typhimurium on beef carcass surfaces. Meat Sci. 69, 401–407. Ko, J., Hamilton, J.A., Ton-Nu, H.T., Schteingart, C.D., Hofmann, A.F., Small, D.M., 1994. Effects of side chain length on ionization behavior and transbilayer transport of unconjugated dihydroxy bile acids: a comparison of nor-chenodeoxycholic acid and chenodeoxycholic acid. J. Lipid Res. 35, 883–892. Kobayashi, H., Oethinger, M., Tuohy, M.J., Hall, G.S., Bauer, T.W., 2009. Improving clinical significance of PCR: use of propidium monoazide to distinguish viable from dead Staphylococcus aureus and Staphylococcus epidermidis. J. Orthop. Res. 27, 1243–1247. Lee, J.-L., Levin, R.E., 2009a. A comparative study of the ability of EMA and PMA to distinguish viable from heat killed mixed bacterial flora from fish fillets. J. Microbiol. Methods 76, 93–96. Lee, J.-L., Levin, R.E., 2009b. Discrimination of viable and dead Vibrio vulnificus after refrigerated and frozen storage using EMA, sodium deoxycholate and real-time PCR. J. Microbiol. Methods 79, 184–188. Løvdal, T., Hovda, M.B., Björkblom, B., Møller, S.G., 2011. Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua. J. Microbiol. Methods 85, 164–169. Nocker, A., Camper, A.K., 2006. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl. Environ. Microbiol. 72, 1997–2004. Nocker, A., Cheung, C.Y., Camper, A.K., 2006. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbiol. Methods 67, 310–320. Nocker, A., Sossa-Fernandez, P., Burr, M.D., Camper, A.K., 2007a. Use of propidium monoazide for live/dead distinction in microbial ecology. Appl. Environ. Microbiol. 73, 5111–5117. Nocker, A., Sossa, K.E., Camper, A.K., 2007b. Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR. J. Microbiol. Methods 70, 252–260. Shi, H., Xu, W., Luo, Y., Chen, L., Liang, Z., Zhou, X., Huang, K., 2011. The effect of various environmental factors on the ethidium monoazide and quantitative PCR method to detect viable bacteria. J. Appl. Microbiol. 111, 1194–1204. Thanassi, D.G., Cheng, L.W., Nikaido, H., 1997. Active efflux of bile salts by Escherichia coli. J. Bacteriol. 179, 2512–2518. USDA, 2013. Safe and suitable ingredients used in the production of meat, poultry, and egg products. Available at: http://www.fsis.usda.gov/OPPDE/rdad/FSISDirectives/ 7120.1.pdf (Accessed August 10, 2013). Yang, X., Badoni, M., Gill, C.O., 2011. Use of propidium monoazide and quantitative PCR for differentiation of viable Escherichia coli from E. coli killed by mild or pasteurizing heat treatments. Food Microbiol. 28, 1478–1482. Yang, X., Badoni, M., Youssef, M.K., Gill, C.O., 2012. Enhanced control of microbiological contamination of product at a large beef packing plant. J. Food Prot. 75, 144–149.