Response of Escherichia coli O157:H7 survival to pH ...

J Soils Sediments DOI 10.1007/s11368-014-0944-y

SOILS, SEC 5 & SOIL AND LANDSCAPE ECOLOGY & RESEARCH ARTICLE

Response of Escherichia coli O157:H7 survival to pH of cultivated soils Haizhen Z. Wang & Gang Wei & Zhiyuan Y. Yao & Jun Lou & Kongcao C. Xiao & Laosheng S. Wu & Jianjun J. Wu & Jianming M. Xu

Received: 24 October 2013 / Accepted: 30 June 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Purpose The Escherichia coli (E. coli) O157:H7 survival dynamics in original and pH-modified agricultural soils were investigated to determinate how E. coli O157:H7 survival responded to the pH values of different soils, identify the relationships between E. coli O157:H7 survival time (td) and soil properties, and assess the potential pathogen contamination after soil pH changed. Materials and methods The six soil samples were collected from different provinces of China, and 18 pH-modified soil samples were obtained from original soils by treating the original soils with direct electric current. The E. coli O157:H7 cells were inoculated into 24 soils and incubated at soil moisture of −33 kPa and 25 °C. The soils were sampled for determining the numbers of E. coli O157:H7 at given time intervals over the incubation. The effects of soil pH change and other properties on the td values were analyzed. Results and discussion The td values in the test soils were between 7.1—24.7 days. Results indicate that soil pH, texture, and free Fe2O 3 (Fed) were the most important factors impacting the td values in the test soils. Further, the response of E. coli O157:H7 survival to pH change varied with different soils. In the acidic soils (shorter td values), the td values decreased as the pH decreased and Fed increased, while in the neutral or alkaline soils (pH≥6.45, longer td values), the td values did not change significantly with pH. Conclusions The changes of amorphous and free sesquioxides induced by pH change might strengthen the response of E. coli Responsible editor: Yanfen Wang H. Z. Wang (*) : G. Wei : Z. Y. Yao : J. Lou : K. C. Xiao : L. S. Wu : J. J. Wu : J. M. Xu (*) Institute of Soil and Water Resources and Environmental Science, Zhejiang Provincial Key Laboratory of Subtropical Soil and Plant Nutrition, Zhejiang University, Hangzhou 310058, China e-mail: [email protected] e-mail: [email protected]

O157:H7 survival to soil pH. Closer attention should be paid to E. coli O157:H7 long survival in soils and its potential environmental contamination risk. Keywords Escherichia coli O157:H7 . Survival . Soil pH . Clay . free Fe2O3

1 Introduction Animal manure and sewage sludge are routinely applied to croplands as fertilizer or as soil amendment all over the world. For instances, Ogden et al. (2009) reported that the annual amount of animal manure applied to land was estimated at 4.3×105 t dry weight in the UK. In 2009, the dewatered sewage sludge was about 3.8×106 t in China, of which approximately 45 % were applied to croplands (Chen et al. 2012). A variety of pathogenic bacteria, including Escherichia coli O157:H7 (E. coli O157:H7), Salmonella, and Campylobacter were found in animal manure or sewage sludge. These pathogenic microorganisms can enter the surface water, irrigation water, soil, and fresh produce, and become a reservoir for future infections (Solomon et al. 2002; Patel et al. 2010; Bradford et al. 2013; Drozd et al. 2013). E. coli O157:H7, an important pathogen, associated with several foodborne and waterborne outbreaks of gastrointestinal illness, has been widely reported in China and other countries around the world in the last decades (Banatvala et al. 2001; Liu et al. 2007; Drozd et al. 2013). Therefore, it is critical to study E. coli O157:H7 survival in environment in order to reduce its potential contamination risk. E. coli O157:H7 has been shown to survive in soil for days to more than 1 year following the application of contaminated water or compost (Jiang et al. 2002; Franz et al. 2008; Patel et al. 2010; Bradford et al. 2013). Most of the previous studies have shown that soil pH is one of the dominant factors

J Soils Sediments

affecting E. coli O157:H7 survival in soil (Jiang et al. 2002; Franz et al. 2008; Ma et al. 2012; Yao et al. 2013; Zhang et al. 2013; Wang et al. 2014). Analysis of these mentioned reports indicates that E. coli O157:H7 survival times (td) are usually longer in alkaline or neutral soils than in acidic soils, or the td values increase with soil pH. However, it is interesting to note that the long td values were negatively or insignificantly related to soil pH in the neutral or alkaline soils (pH, 6.70–8.31) (Franz et al. 2008; Ma et al. 2012; Yao et al. 2013). Unfortunately, no explanation was provided for the conflicting relation between soil pH and the td values in those studies. Nevertheless, it is an interesting hypothesis that whether or not the effects of soil pH on E. coli O157:H7 survival is varied with different soil types. More importantly, soil pH can be affected by natural process or anthropogenic activities, including land use change, atmospheric acid deposition, and application of excessive ammonium-based fertilizers or irrigation water (Guo et al. 2010; Butterly et al. 2013). For instances, decrease in soil pH due to soil acidification is a serious environmental issue worldwide as it can significantly affect agricultural sustainability (Guo et al. 2010). Meanwhile, soil amendments (e.g., liming, manure, crop residues, biochars, etc.) have also been widely applied to correct soil acidity and enhance agricultural productivity (Pagani and Mallarino 2012; Butterly et al. 2013; Dai et al. 2013). Nevertheless, these studies focused mostly on the dynamics of soil pH and its impacts on soil nutrients availability (Guo et al. 2010; Pagani and Mallarino 2012; Butterly et al. 2013; Dai et al. 2013). There is a dearth of information on how changing in soil pH may affect pathogen survival in soil, while such information is critical to evaluate and control potential environmental contamination risk of the pathogen in natural environment. In this study, six soils with different properties were collected to investigate how E. coli O157:H7 survival responds to pH change in different soils. Since E. coli O157:H7 survival time (td) can be affected more by soil pH than by soil indigenous microorganisms (Ma et al. 2012; Zhang et al. 2013). The aims of this study were to (1) identify the relationships between E. coli O157:H7 survival time and soil physicochemical properties and (2) assess the possible contamination risk of E. coli O157:H7 when soil pH changes.

2 Materials and methods 2.1 Soils Six representative cropland soils of northeast China (S1), North China Plain (S2 and S3), and south China (S4 to S6) were collected from the 0–20 cm layer. Soils S1 to S4 were from four national stations of Agro-Ecology Research: Hailun (S1, Luvic Phaeozems, 47.454° N, 126.928° E, Heilongjiang

Province), Changwu (S2, Cumulic Haplic Kastanozems, 35.241° N, 107.683° E, Shaanxi Province), Fengqiu (S3, Calcaric Fluvisols, 35.011° N, 114.329° E, Henan Province), and Qiyang (S4, Plinthic Acrisols, 26.761° N, 111.876° E, Hunan Province), respectively. Soils S5 (Umbric Acrisols, 30.667° N, 120.733° E) and S6 (Haplic Acrisols, 29.019° N, 119.467° E) were from Zhejiang Province. Three replicates were sampled at each field site, and each sample was a composite of several individual soil cores taken along a 200m transect. The fresh samples were sieved to pass through a 2mm plastic mesh, air-dried, and homogenized thoroughly. A sub-sample was collected from each sample for analyzing physical and chemical properties. The six original soils have different pH values and other properties (Table 1). In order to evaluate the effect of pH change on E. coli O157:H7 EDL93 survival in the soils, soil pH gradients of each original soil were obtained by the application of direct electric current (DEC) based on the theory that the electrolysis of water occurs at the anode: 2H2O → 4H+ + O2 + 4e− and at the cathode: 2H2O + 2e− → 2OH− + H2, which causes pH increase in the soil region near the cathode, while pH decrease in the soil region near the anode, resulting in a pH gradient between the electrodes (Saichek and Reddy 2003). Experimentally, an electrokinetic apparatus similar to the method by Lear et al. (2004) was used. It consists of a DEC power supply, a reactor, two electrode chambers, and a pump. The electrokinetic reactor was made from PVC material and had a soil compartment (50×20×10 cm) and two electrode chambers (2×20×10 cm). Each of the electrode chambers contained four compressed plate-type graphite electrodes (5×3×0.8 cm). The electrokinetic compartment was packed with air-dried soil to a depth of 6 cm and subsequently saturated with water and allowed to equilibrate for 24 h prior to DEC application, then a constant voltage gradient of 1 V cm−1 was applied to the electrode. Forty eight hours after the electrokinetic process, the soil was divided into seven sections by the distance from anode to cathode: 0–5, 5–13, 13–21, 21–29, 29–37, 37–45, and 45–50 cm. Since the significant impact of DEC on soil properties was only observed in the soil close to the anode or cathode (Pazos et al. 2012), subsamples in the two end sections (0–5 and 45– 50 cm) were excluded in our study. The subsamples in other five sections were removed from the compartment, air-dried, and sieved (2 mm). Based on the study by Guo et al. (2010), the pH values of soils in cereal crop systems in northeast China (e.g., S1), north China Plain (e.g., S2 and S3), and south China (e.g., S4–S6) were 4.84–7.60, 5.37–8.70, and 4.17–6.52, respectively, we selected three sections (subsamples) of each DEC-treated soil to match the above pH ranges with a pH gradient of about 0.5 unit as much as possible. Together with the original soil sample (without treated with DEC), each soil type consisted of a set of four

J Soils Sediments Table 1 The selected properties of the test soils Fed

Alo

Ald

Soil code

pH

ZP mV

OC g kg−1

TN

WSOC mg kg−1

AK

clay %

sand

Feo g kg−1

S1A S1 S1B S1C

5.29 5.72 6.45 6.68

−26.1 −26.2 −26.0 −25.7

38.2 36.7 35.8 36.1

3.7 4.0 3.9 3.9

279.1 277.2 271.1 266.0

195.4 193.6 194.8 189.6

39.0 39.2 39.4 39.4

15.0 15.4 15.2 15.0

4.1 4.1 4.3 3.9

6.9 6.5 6.1 4.8

4.5 4.3 4.1 4.0

4.6 3.3 3.2 2.5

S2A S2 S2B S2C S3A S3 S3B S3C S4 S4A S4B S4C S5A S5B S5 S5C S6A S6B

7.84 8.13 8.55 8.91 7.73 8.28 8.72 9.16 4.30 4.60 4.94 5.34 4.14 4.75 5.41 5.77 4.49 5.07

−20.3 −20.5 −20.0 −20.7 −20.8 −18.9 −19.4 −20.0 −22.4 −21.7 −23.1 −23.2 −24.0 −23.6 −23.2 −22.4 −22.5 −23.9

7.5 7.9 7.0 7.0 8.5 8.5 8.6 8.6 8.5 8.5 8.4 8.1 17.7 17.6 18.1 17.2 10.7 10.9

1.9 1.9 1.8 1.9 1.8 1.7 1.7 1.7 1.8 1.8 1.8 1.8 3.4 3.5 3.8 3.9 2.2 2.2

214.1 216.6 201.9 210.3 262.8 256.1 268.9 255.4 80.7 75.0 70.7 86.1 269.2 264.8 246.8 253.3 140.9 136.3

209.0 192.8 206.6 220.1 50.9 62.1 56.5 72.3 93.6 98.3 96.8 98.6 118.8 128.0 138.1 135.0 126.4 129.1

27.5 27.3 27.7 27.5 16.5 16.1 16.1 16.3 41.0 40.8 41.0 41.0 28.6 28.4 28.2 28.6 33.0 33.0

8.5 9.3 9.1 9.1 15.5 15.9 15.5 15.5 10.0 10.0 9.8 10.2 7.0 7.2 7.0 7.0 16.0 15.8

1.1 0.8 0.9 0.8 1.0 0.8 0.8 0.8 1.4 1.3 1.2 1.2 5.8 5.8 5.7 5.3 2.1 2.0

7.5 6.5 6.6 6.7 4.8 4.0 3.2 3.1 20.5 21.4 21.3 21.1 13.5 13.3 12.7 12.6 25.4 22.1

2.4 2.2 2.2 2.1 1.9 1.7 1.4 1.3 3.3 3.3 3.4 3.2 4.5 4.2 2.8 2.7 3.6 3.6

1.6 0.9 1.0 1.1 0.4 0.2 0.2 0.2 6.4 7.4 7.6 7.3 4.5 3.3 1.6 1.7 12.8 12.0

S6 S6C

5.45 5.98

−23.9 −24.8

10.7 11.4

2.2 2.1

144.2 130.0

137.1 137.9

33.0 33.2

16.0 16.0

2.0 1.7

18.4 17.4

3.5 3.5

9.8 9.5

S1–S6 represent Luvic Phaeozems, Cumulic Haplic Kastanozems, Calcaric Fluvisols, Plinthic Acrisols, Umbric Acrisols, and Haplic Acrisols soils from different provinces of China, respectively; the soil samples suffix with A, B, or C after soil pH modified; ZP zeta potential; OC organic carbon; TN total nitrogen; WSOC water soluble organic carbon; AK available potassium; Feo amorphous Fe2O3; Fed free Fe2O3; Alo amorphous Al2O3; Ald free Al2O3

samples (a set of soil includes the original soil, plus the three DEC-treated soils of different pH values), resulting in a total of six sets, 24 individual samples (Table 1). Selected properties of the DEC-treated soils are shown in Table 1. Briefly, soil pH, organic carbon (OC), available potassium (AK), and texture were tested according to the protocols by Agricultural Chemistry Committee of China (1983). Zeta potential was determined using a ZetaSizer Nano ZS90 (Malvern, Worcs, UK) equipped with zeta cells (DTS1060c; Malvern, Worcs, UK) at 25ºC on 100 mg samples that had been shaken in 60 ml of deionised water (Churchman 2002). Total nitrogen (TN) was determined using a Kjeldahl apparatus (BUCHI KjelFlex K-360; -BÜCHI Labortechnik AG, Flawil, Switzerland), and water-soluble organic carbon (WSOC) was measured by a Multi N/C TOC analyzer (Analytic Jena AG, Jena, Germany). The amorphous and free sesquioxides were extracted with a dithionite-citrate solution buffered with NH4-oxalate (pH 3.0) and NaHCO3 solution, respectively, in the dark (Mehfa and Jackson 1960). Soil moisture at −33 kPa was determined using a pressure membrane

apparatus (SoilMoisture Equipment Corp, Santa Barbara, CA, USA) as described by Richards (1949). 2.2 Incubation experiments Since the strains of E. coli O157:H7 isolated from the outbreaks in China were almost identical with E. coli O157:H7 EDL933 (Liu et al. 2007), E. coli O157:H7 EDL933 was selected as a representative strain in this study. To facilitate the plating analysis of E. coli O157:H7 on the SMAC (sorbitol MacConkey)-BCIG (5-bromo-4-chloro-3-indoxyl-β-D-glucuronide) agar (Lab M, Lancashire, UK), the test E. coli O157:H7 strain was induced to be resistant to 100 μg mL−1 of rifampicin (Fisher Scientific, Fair Lawn, NJ, USA) and 25 μg mL−1 of nalidixic acid (SigmaAldrich, MO, USA) (Wang et al. 2014). Each air-dried soil was adjusted to 80 % of the field capacity water contents (at −33 kPa) by adding sterile deionized water. The soil samples were thoroughly mixed, and after the added water eventually distributed in soil samples, E. coli O157:H7 suspension in sterile deionized water was inoculated

J Soils Sediments

into the soil to establish a cell density of about 3×107 colonyforming units per gram oven-dried weight of soil (CFU g−1). Soil moisture at −33 kPa was generally used to simulate field conditions since it represents the water holding capacity of the soil and the highest available water contents in soil (Richards 1949). Hence, the inoculated soil samples were finally adjusted to their respective field capacity water contents (at −33 kPa), thoroughly mixed again, and placed in a 50-mL sterilized plastic tube. Our previous experiments showed that the plastic tube itself did not affect E. coli O157:H7 survival. The same amount of non-inoculated soil (50 g in oven-dried weight), to which sterile deionized water had been added instead of cell suspension, was put into another tube to serve as uninoculated control. All tubes (three replicates for each soil, plus uninoculated samples) were incubated in the dark at 25±1 °C and sampled at intervals over 25 days (Section 2.3). Sterile deionized water was added to the samples every 2 days as required to maintain quasi-constant soil moisture status at their respective field capacities. 2.3 Sampling and plating analyses Approximately 0.5 g (equivalent to oven-dried weight) of soil was removed from each tube at 0, 0.25, 1, 2, 3, 5, 7, 10, 15, 20, and 25 days after inoculation (DAI). Each sampled soil was thoroughly mixed with 4.5 mL of 0.1 % peptone buffer (Lab M, Lancashire, UK) by inverting the tube several times and then vortexed for 2×30 s. The resulting soil suspension was then subjected to 10-fold serial dilutions. Finally, 0.1 mL of the last three of the serial dilutions per sample was plated onto SMAC-BCIG agar containing 100 μg mL−1 rifampicin and 25 μg mL−1 nalidixic acid, and incubated at 37 °C (16 h) for counting E. coli O157:H7 numbers. The plating analyses were done on each of three replicates of the inoculated and uninoculated samples. Each replicate had three platings, and each replicate-average was calculated from the three platings. The soil (site) averages and error bars of E. coli O157:H7 counts were calculated from three replicateaverages. In this study, the detection limit of the plating method was 100 CFU g−1. The cell recovery rate of the method was with a mean±standard deviation value of 95.0±8.5 % at 0 DAI. Sampling was stopped after plate counts of zero appeared twice in succession during the incubation. For the uninoculated soil samples, there was no E. coli O157:H7 detected by plating analysis during the entire incubation period. Namely, the plating analyses indeed represented the net colonies of the inoculated E. coli O157:H7 reproduction and die-off. 2.4 Data analysis Bacterial populations were converted to log10 (CFU g−1) before statistical analysis. The data were analyzed by the

Weibull survival model [Eq. (1)] proposed by Mafart et al. (2002) using GInaFiT V1.6 developed by Geeraerd et al. (2005): t p ð1Þ log10 ðN t Þ ¼ log10 ðN 0 Þ δ where Nt represents the number of surviving cells remaining at time t, N0 is the initial size of the inoculums population, p is a shape parameter, and δ is a scale parameter that represents the time needed for first decimal reduction; td represents the time when Nt reaches detection limit (100 CFU g−1), which can also be calculated from Eq. (1). In addition, simple correlation analysis, stepwise multiplelinear regression analysis, and path analysis were performed by using SPSS 18.0 for Windows (SPSS Inc., IL, USA) to better understand how soil properties affected E. coli O157:H7 survival time (td). Path coefficients (PC) and determinative coefficients (DC) were also calculated to assess the direct and indirect effects of soil properties on the td values following the method by Wright (1934). Analysis of variance (ANOVA) was carried out to test the differences at 5 % significant level of the p, δ, and td values among soils by SPSS statistic software. Multiple factor analysis (MFA), which developed by Escofier and Pagès (1982), was applied to further visualize E. coli O157:H7 survival and its relationship with soil properties by using R software FactoMineR package (Husson et al. 2013; R Core Team 2013). Finally, MFA provided a correlation circle of two groups of variables [i.e., survival parameters (p, δ, and td) and the selected soil properties] and a superimposed representation of the test soils described by each group of variables in the principal plane. Each soil sample was represented by three aligned points: two partial points (i.e., described by only one group) and a mean point (in the centroid of the two previous ones).

3 Results 3.1 Survival dynamics of E. coli O157:H7 in soils Results indicated that the level of inoculated E. coli O157:H7 colonies in soil samples decreased with time over the incubation period (Fig. 1). However, E. coli O157:H7 survival dynamics varied among the test soils. The colonies declined slowly and survived about 20 to 25 days before reaching the detection limit (100 CFU g−1) in soil sets S2 and S3. Rather, E. coli O157:H7 declined rapidly in soil sets S4–S6 (Fig. 1). E. coli O157:H7 survival data fitted well to the Weibull model (Eq. 1) with R2 ranging from 0.894 to 0.995 for all the test soils. The calculated average td values of E. coli O157:H7 in the test soils were from 7.1 to 24.7 days. The p, δ, and td values were generally higher in soil sets S1–S3 than those in soil sets S4–S6 (Fig. 2). Moreover, the effect of soil pH changes on E. coli O157:H7 survival was varied in different soils (Figs. 1 and 2). It was

J Soils Sediments

6

4

2

0

5

10

15

20

6

4

2

0

25

S2A S2 S2B S2C

0

5

10

Time (day)

6

4

2

5

10 15 Time (day)

20

25

8

CFU counts (log10CFU g-1))

CFU counts (log10(CFU g-1))

S4 S4A S4B S4C

0

20

6

4

2

0

25

S3A S3 S3B S3C

0

5

Time (day)

8

0

15

8

S5A S5B S5 S5C

6

4

2

0

0

5

10 15 Time (day)

20

25

10 15 Time (day)

20

8


0

8


S1A S1 S1B S1C



8

25

S6A S6B S6 S6C

6

4

2

0

0

5

10 15 Time (day)

20

25

Fig. 1 Survival dynamics of E. coli O157:H7 in the test soils. Bars are ± the standard deviation of means of three replicates; CFU g−1 is the colonyforming units per gram of dry soil. The soil codes are the same as shown in Table 1

especially obvious in soil S1 from Heilongjiang Province. E. coli O157:H7 survival times (td) in the original soil S1 were about 13 days, but about 22 days in S1B and S1C with higher pH. Test of homogeneity of variances showed that no significant differences were present in the variances of p (P, 0.09–0.77), δ (P, 0.07–0.35), and td (P, 0.07–0.74) among different soil pH values in each of the soil samples, thus all samples were pooled to carried out ANOVA. ANOVA indicated that there were differences in the parameters of E. coli O157:H7 survival model (p, δ, and td) due to pH change of the test soils (Fig. 2). For instances, compared with the original soils, the td values decreased in the acidic soils S5 and S6 from Zhejiang province after their pH values were modified to lower than the original soils. However, pH change did not significantly impact the td values in alkaline soil samples from Shaanxi (S2) and Henan (S3) provinces. 3.2 Relationship between soil property and E. coli O157:H7 survival in the original soils Simple correlation analysis revealed that td was positively correlated with pH (r=0.952, P>clay content> sand content (td =−29.624+5.707×pH+0.364×clay−0.233× sand, R2 =0.998). Both coefficients of multiple regression and partial correlation were significant at PS3, S5>S2, and S6>S4 in this study (Fig. 4), which agreed with the findings in Figs. 1 and 2 and Table 1. Further examination of Figs. 3 and 4 revealed that the data points of soil sets S4–S6 were close on the left of the axis (Fig. 4), which corresponding to low pH, short td values, and high Fed content (Fig. 3). On the other hand, soil sets S1–S3 were on the right of the axis (Fig. 4) corresponding to high pH and td values and low Fed content (Fig. 3). Separation of data points of soil set S1 from those of soil sets S2 and S3 was attributed to the highest OC content and variable td values in soil set S1.

Fig. 2 The Weibull model parameters (p and δ) and survival time (td, days) for E. coli O157:H7 survival in the test soils. Bars are ± the standard deviation of means of three replicates. Without the same lower-case letter marked above the columns indicates significant difference among the test soils at the 0.05 probability level (P