Interaction between the Microbial Community and InvadingEscherichia coli O157:H7 in Soils from Vegetable Fields

Zhiyuan Yao, Haizhen Wang, Laosheng Wu, Jianjun Wu, Philip C. Brookes, Jianming Xu

‹Institute of Soil and Water Resources and Environmental Science, Zhejiang Provincial Key Laboratory of Subtropical Soil and Plant Nutrition, Zhejiang University,Hangzhou, China

The survival of Escherichia coli O157:H7 in soils can contaminate vegetables, fruits, drinking water, etc. However, data on theimpact of E. coli O157:H7 on soil microbial communities are limited. In this study, we monitored the changes in the indigenousmicrobial community by using the phospholipid fatty acid (PLFA) method to investigate the interaction of the soil microbialcommunity with E. coli O157:H7 in soils. Simple correlation analysis showed that the survival of E. coli O157:H7 in the test soilswas negatively correlated with the ratio of Gram-negative (G�) to Gram-positive (G�) bacterial PLFAs (G�/G� ratio). In particu-lar, levels of 14 PLFAs were negatively correlated with the survival time of E. coli O157:H7. The contents of actinomycetous andfungal PLFAs in the test soils declined significantly (P, <0.05) after 25 days of incubation with E. coli O157:H7. The G�/G� ratiodeclined slightly, while the ratio of bacterial to fungal PLFAs (B/F ratio) and the ratio of normal saturated PLFAs to monounsat-urated PLFAs (S/M ratio) increased, after E. coli O157:H7 inoculation. Principal component analysis results further indicatedthat invasion by E. coli O157:H7 had some effects on the soil microbial community. Our data revealed that the toxicity of E. coliO157:H7 presents not only in its pathogenicity but also in its effect on soil microecology. Hence, close attention should be paidto the survival of E. coli O157:H7 and its potential for contaminating soils.

Soil pollution by heavy metals or organic pollutants has beenstudied extensively in the past few decades. Many researchers

have reported that soil microbial communities respond to thepresence of heavy metals and organic pollutants (1–6). Increasingnumbers of studies in recent years have also been conducted onsoil biopollution by pathogenic microorganisms, including vi-ruses, protozoa, and bacteria such as Escherichia coli O157:H7,Salmonella spp., and Listeria monocytogenes (7–9). E. coli O157:H7, a significant pathogen carried naturally by animals, has beenshown to pose a significant threat to environmental safety andpublic health because of its low infective dose (as few as 10 cells)and its high pathogenicity in watery diarrhea, hemorrhagic colitis,hemorrhagic-uremic syndrome, thrombotic thrombocytopenicpurpura, etc. (10). This organism has been placed on the list ofpotential bioterrorism agents by the U.S. government, followed bymany nations and international bodies (11). Hence, assessment ofthe potential contamination risk posed by E. coli O157:H7 in theenvironment is critically important.

Manure-borne zoonotic pathogens can invade the soil throughsewage irrigation, runoff from stored manure, application of solidmanure to fields, or other processes (12–15). Invading E. coliO157:H7 has been observed to survive in soil or soil-related (ma-nure) environments for days to more than 1 year (9, 15–19). Thefate of E. coli O157:H7 in soil can be affected by both biotic andabiotic factors (9, 12, 15). Our previous study revealed that the soilmicrobial community, especially bacteria and fungi, played animportant role in the survival of E. coli O157:H7 (9), a findingconsistent with those of other studies (15, 19, 20). The decline inmicrobial diversity coincided with an enhancement of the survivalrate of invading E. coli O157:H7 (17, 20). Apart from the diversityof indigenous microflora in the soil, certain groups of microor-ganisms might also contribute to the persistence of E. coliO157:H7 in soil (19). However, we still lack sufficient understand-ing of how the diversity and community composition of soil mi-crobial communities influence the survival of E. coli O157:H7 in

soil. In addition, little information is available on the impact of E.coli O157:H7 on microorganisms indigenous to the soil.

Numerous reports have indicated that contamination withheavy metals and organic pollutants exerts a negative influence onsoil biological properties such as microbial biomass, enzyme ac-tivity, and microbial diversity (1, 2, 4, 5, 21). Furthermore, micro-organisms are often used in agriculture and horticulture to im-prove soil fertility (22), to promote plant growth (23), or tofacilitate the bioremediation of polluted sites (24). The microor-ganisms introduced will compete with microorganisms indige-nous to the soil for nutrients and niche space (25). Studies haveshown that bioaugmentation through the introduction of micro-organisms can change the structure of the soil microbial commu-nity and even disrupt ecological balance (22, 26, 27). With regardto pathogenicity, most of the research has focused on the survivaland transport of E. coli O157:H7 in soil, while the potential effectof E. coli O157:H7 on the microbial community in agricultural soilis still unknown. Thus, it is important to address the displacementof indigenous microorganisms by inoculants and its potential ef-fects on the soil microcosm (28).

Phospholipid fatty acids (PLFAs) are the specific componentsof cell membranes that are found only in intact (viable) cells (29).Many organisms produce specific or signature types of PLFA bio-markers, which can then provide a fingerprint of soil microbiota(2–4, 30, 31). To date, PLFAs have been widely used to exploreenvironmental effects on the soil microbial community (2, 3, 5,

Received 10 September 2013 Accepted 7 October 2013

Published ahead of print 11 October 2013

Address correspondence to Haizhen Wang, mywhz@163.com, or Jianming Xu,jmxu@zju.edu.cn.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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11). Hence, detailed information about the effect of E. coliO157:H7 invasion on the soil microbial community can be ob-tained via PLFA analysis. In this study, PLFA analysis was used toinvestigate the interaction between the invading E. coli O157:H7and the soil microbial community. More specifically, the aims ofthis research were (i) to determine the effects of different micro-bial groups among the indigenous microorganisms on the survivalof E. coli O157:H7 in soils from vegetable fields under plastic-greenhouse cultivation for various lengths of time (0, 1, 4, 7, and15 years), (ii) to identify the changes in the soil microbial commu-nity induced by invading E. coli O157:H7, and (iii) to providecomprehensive information for the evaluation and control of thesurvival of this pathogen in the natural environment.

MATERIALS AND METHODSSoil collection and analysis. The soil samples used in this study (S0, S1,S4, S7, and S15) were from fields under plastic-greenhouse cultivation forvarious lengths of time (0, 1, 4, 7, and 15 years, respectively) in ShandongProvince, China, and were collected from the surface horizon (0 to 20 cm)(9). The samples were of the same soil type, and the sampling sites sharedsimilar weather conditions, including mean annual temperature (13.1°C)and precipitation (578.4 mm), due to the closeness of their locations. Atotal of 15 plots were sampled in this study, triplicate plots for each plastic-greenhouse cultivation period. The soil sample from each plot was a com-posite of 5 individual soil cores taken at 5-m intervals. Bagged compositesamples were taken to the laboratory using coolers filled with ice. Leaves,roots, and stones were manually removed before the soil samples weresieved (pore size, �2 mm) and stored at 4°C. For each composite soilsample, a portion of sieved subsample was freeze-dried and was thensubjected to PLFA analysis according to the method described by He et al.(3). The individual PLFAs of the test soils are shown in Table 1, and othersoil properties (pH, water-soluble organic carbon, available phosphorus,available potassium, organic carbon, total nitrogen, field capacity [soil

water content at �33 kPa], clay, total PLFAs, bacterial PLFAs, and fungalPLFAs) have been reported previously by Yao et al. (9).

Incubation experiments. Before the inoculation experiment, the soilsamples, previously stored at 4°C, were incubated in a darkroom at 25 �1°C for 5 days to activate the soil microbial communities (9, 32). E. coliO157:H7 derived from E. coli O157:H7 EDL933 (ATCC 43895) was usedas the test bacterial strain, as described previously by Yao et al. (9). Thefinal cell density of the bacterial suspension was determined by serialdilution and plate colony counting. The E. coli O157:H7 suspension wasinoculated into the soil to establish an inoculation density of about 106

CFU per g (oven-dry weight) of soil. Forty grams (oven-dry weight) of theinoculated soil was placed in a 50-ml sterile plastic tube. For the uninocu-lated control, the same amount of uninoculated soil was put into anothertube, and sterilized deionized water was added instead of the cell suspen-sion. All samples (triplicates both for each treatment and for uninoculatedcontrols) were incubated in the dark at 25 � 1°C with the field capacity(soil water content at �33 kPa). Moisture loss during incubation wascompensated for by adding sterile deionized water every 2 days to main-tain a constant soil moisture status.

Survival data modeling. At 0, 0.04, 1, 3, 5, 7, 10, 15, and 25 days afterinoculation, approximately 0.5 g (oven-dry weight) of soil sample wastaken from each tube to determine the survival of E. coli O157:H7 overtime (9). The experimental data were fitted to the Weibull survival func-tion by the following equation, as described by Wang et al. (32): log10(Nt) �log10(N0) � (t/�)p, where Nt represents the number of surviving cellsremaining at time t, N0 is the inoculum size, p is the shape parameter, and� is the scale parameter that represents the time (day) needed for firstdecimal reduction (32–34). The time when Nt reaches the detection limit(100 CFU · g�1), td, can also be calculated from this equation. There are 3data sets for the survival of E. coli O157:H7 in each soil sample (S0, S1, S4,S7, and S15) in the study. The average td values of E. coli O157:H7 for thetest soils ranged from 17.92 to 22.64 days (9) (Table 1, footnote).

PLFA analysis. Soil samples were taken from the inoculated and uni-noculated soils after 25 days of incubation and were subjected to PLFAanalysis as described by He et al. (3). Following methylation of the polarlipids, PLFA methyl esters were separated and identified by gas chroma-tography (Agilent 6890N gas chromatograph; Agilent, Wilmington, DE,USA) fitted with a MIDI peak identification system (version 4.5; MIDI,Newark, NJ, USA). The 19:00 fatty acid was added as an internal standardbefore methylation. For each sample, the concentration of individual fattyacid methyl esters is expressed as nanomoles of PLFA per gram (oven-dryweight) of soil. Individual fatty acids are designated in terms of the totalnumber of carbon atoms and the number of double bonds, followed bythe position (�) of the double bond from the methyl end of the molecule(30, 31). The prefixes “a” and “i” describe anteiso and iso branching,respectively; “10Me” represents a methyl group on the 10th carbon atomfrom the carboxyl end of the molecule; and “cy” indicates a cyclopropanefatty acid (31). The suffix “c” indicates a cis-fatty acid (30). The PLFAbiomarkers associated with the different PLFA groups, including bacte-rial, Gram-negative (G�) bacterial, Gram-positive (G�) bacterial, actino-mycetous, fungal, branched, iso, anteiso, cyclopropyl, saturated, andmonounsaturated PLFAs, were determined by previous studies (2–5, 30)(Table 2). PLFAs present at concentrations of �0.1 nmol · g�1 or PLFAsthat were observed in only one sample were eliminated from the data set.Finally, 21 PLFAs existing in each soil sample were used for statisticalanalysis.

Statistical analysis. The PLFA data set was subjected to principal com-ponent analysis (PCA) in order to examine the change in the soil micro-bial community after incubation with E. coli O157:H7. PLFA profiles inthe fresh soil samples were regarded as the biological properties of soils.The relationships between biological properties (each individual PLFA,signature microbial groups, and particular PLFA ratios) and E. coliO157:H7 survival time (td) were evaluated by the correlation coefficient(r). The r values in Table 2 were calculated with the td data and the corre-sponding concentrations of individual PLFAs across the 15 plots sampled.

TABLE 1 Concentrations of individual phospholipid fatty acids in freshsoils

PLFA

Concn (nmol · g�1) of PLFA in the following soil samplea:

S0 S1 S4 S7 S15

15:00 0.44 � 0.03 0.26 � 0.03 0.47 � 0.09 0.47 � 0.05 0.40 � 0.0117:00 0.67 � 0.11 0.45 � 0.05 0.68 � 0.09 0.68 � 0.02 0.60 � 0.02a15:0 2.98 � 0.09 1.43 � 0.10 2.41 � 0.15 2.84 � 0.15 2.09 � 0.13a17:0 2.01 � 0.26 1.57 � 0.14 1.92 � 0.31 1.95 � 0.27 1.59 � 0.23i14:0 0.53 � 0.10 0.17 � 0.02 0.31 � 0.05 0.39 � 0.03 0.33 � 0.06i15:0 5.00 � 0.62 2.54 � 0.26 3.96 � 0.51 5.00 � 0.23 3.91 � 0.02i16:0 2.34 � 0.15 1.22 � 0.07 1.61 � 0.07 1.97 � 0.10 1.57 � 0.08i17:0 2.04 � 0.08 1.20 � 0.08 1.78 � 0.07 1.98 � 0.09 1.74 � 0.20i18:0 0.00 � 0.00 0.25 � 0.05 0.38 � 0.06 0.32 � 0.02 0.37 � 0.08cy17:0 2.65 � 0.18 1.29 � 0.20 1.85 � 0.12 2.08 � 0.06 1.48 � 0.00cy19:0 8.21 � 1.08 4.31 � 0.46 5.93 � 0.49 5.90 � 1.00 5.50 � 0.6210Me16:0 8.20 � 1.69 4.03 � 0.52 6.62 � 1.09 7.63 � 0.82 5.21 � 1.0710Me17:0 0.61 � 0.06 0.29 � 0.03 0.33 � 0.04 0.36 � 0.05 0.32 � 0.0410Me19:0 0.39 � 0.07 0.24 � 0.12 0.25 � 0.05 0.27 � 0.02 0.20 � 0.0016:1�5c 3.81 � 0.25 1.53 � 0.06 2.37 � 0.02 3.11 � 0.11 2.10 � 0.1716:1�7c 6.21 � 0.75 3.20 � 0.17 4.20 � 0.12 5.12 � 0.61 4.07 � 0.3017:1�8c 0.90 � 0.23 0.70 � 0.13 0.66 � 0.05 0.75 � 0.06 0.62 � 0.0318:1�7c 11.61 � 1.49 5.07 � 0.26 7.65 � 0.11 7.87 � 0.73 6.35 � 0.5618:1�9c 4.30 � 0.85 2.44 � 0.16 3.19 � 0.32 3.12 � 0.45 2.92 � 0.6418:2�6,9c 2.07 � 0.08 0.99 � 0.15 1.51 � 0.18 2.08 � 0.24 1.11 � 0.0820:1�9c 2.82 � 0.12 1.40 � 0.16 2.12 � 0.17 2.72 � 0.38 1.63 � 0.02

a S0, S1, S4, S7, and S15 designate vegetable soils under plastic-greenhouse cultivationfor various lengths of time (0, 1, 4, 7, and 15 years, respectively). The survival time of E.coli O157:H7 until it reaches the detection limit (100 CFU · g�1) is 17.92 � 0.07 daysfor S0, 22.17 � 0.25 days for S1, 20.39 � 0.25 days for S4, 22.62 � 0.04 days for S7, and22.51 � 0.04 days for S15.

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Redundancy analysis (RDA), a linear canonical community ordinationmethod, was performed by using CANOCO for Windows, version 4.02(35), to visualize the relationships between the soil microbial communityand td. All other analyses were carried out by SPSS, version 18.0 for Win-dows (SPSS Inc., Chicago, IL, USA).

RESULTSPhospholipid fatty acid analysis. Fifty-six PLFAs, including bac-terial, fungal, actinomycetous, branched, anteiso, cyclopropyl,iso, and monounsaturated fatty acids, were detected in the testsoils. Among these, 21 PLFAs with chain lengths ranging from 14to 20 carbon atoms were identified and were reported as the bio-markers of different PLFA groups (Table 2). All of the 21 distinctPLFAs were detected in the test soils irrespective of E. coliO157:H7 inoculation. However, the PLFA profiles of the differenttest soils differed (Table 1; Fig. 1 and 2). The average concentra-tions of individual PLFAs in the test soils ranged from 0.26nmol·g�1 (i18:0) to 7.71 nmol·g�1 (18:1�7c); the i18:0 and 18:1�7c PLFAs belong to iso-PLFAs and G� bacterial PLFAs, respec-tively (Table 2). The microbial biomass, expressed as total PLFAs,ranged widely from 38.73 nmol · g�1 to 91.88 nmol · g�1 in thetest soils (Fig. 1). For the fresh soils, the highest total-PLFAconcentrations were found in open-field soil (S0), as was the case forthe bacterial, G� bacterial, G� bacterial, actinomycetous, and fungalPLFAs (Fig. 1). The contents of signature microbial groups dif-fered from each other, with a trend for bacterial PLFAs to be mostnumerous, followed, in descending order, by G� bacterial PLFAs,G� bacterial PLFAs, and actinomycetous and fungal PLFAs(Fig. 1).

TABLE 2 Main phospholipid fatty acid biomarkers studied and theircorrelations with E. coli O157:H7 survival time

PLFAbiomarker

PLFA groupa

rbB G� G� A F Br I AI Cy S M

15:00 �0.28217:00 �0.370a15:0 �0.517*a17:0 �0.382i14:0 �0.606*i15:0 �0.400i16:0 �0.644**i17:0 �0.407i18:0 0.758**cy17:0 �0.761**cy19:0 �0.736**10Me16:0 �0.48310Me17:0 �0.811**10Me19:0 �0.704**16:1�5c �0.652**16:1�7c �0.632*17:1�8c �0.519*18:1�7c �0.812**18:1�9c �0.682**18:2�6,9c �0.46620:1�9c �0.514*a Check marks indicate which PLFA biomarkers correspond to which PLFA groups.Abbreviations stand for bacterial (B), Gram-negative bacterial (G�), Gram-positivebacterial (G�), actinomycetous (A), fungal (F), branched (Br), iso (I), anteiso (AI),cyclopropyl (Cy), saturated (S), and monounsaturated (M) PLFAs.b r, coefficient of correlation between the individual PLFA and the E. coli O157:H7survival time (td) across the 15 plots sampled. Asterisks indicate significant correlations(*, P � 0.05; **, P � 0.01).

FIG 1 Total phospholipid fatty acids (PLFAs) and PLFA biomarkers of specific microbial groups in the test soils. Data are means � 1 standard deviation for threereplicates. C, fresh soil samples; W, soils incubated without E. coli O157:H7 for 25 days; E, soils incubated with E. coli O157:H7 for 25 days. The same lowercaseletters indicate no significant differences, and different lowercase letters indicate significant differences (P � 0.05), among C, W, and E. G� PLFAs, Gram-negative bacterial PLFAs; G� PLFAs, Gram-positive bacterial PLFAs. The soil codes (S0, S1, S4, S7, and S15) are explained in the footnote to Table 1.

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Effect of soil microbial community on E. coli O157:H7 sur-vival. The results of simple correlation analysis showed thatamong the 21 individual PLFAs identified, 14 individual PLFAswere related to the survival time of E. coli O157:H7 (td). Themonounsaturated fatty acids (16:1�5c, 16:1�7c, 17:1�8c, 18:1�7c, 18:1�9c, and 20:1�9c) and the cyclopropane fatty acids(cy17:0, cy19:0) were all negatively correlated with the td of E. coliO157:H7 at a significance level (P) of �0.05, as were the iso (i14:0,i16:0), anteiso (a15:0), and methyl group (10Me17:0, 10Me19:0)PLFAs (Table 2). Moreover, significantly negative correlationswere found between td and total PLFAs (r, �0.698; P, �0.01),bacterial PLFAs (r, �0.678; P, �0.01), fungal PLFAs (r, �0.654;P, �0.01), and the ratio of G� bacterial PLFAs to G� bacterial PLFAs(G�/G�) (r, �0.698; P, �0.01). Redundancy analysis (RDA) wasused to further explore the influence of soil microbial communi-ties on the survival time of E. coli O157:H7 in test soils (Fig. 3).This analysis confirmed that the ratio of bacterial to fungal PLFAs(B/F) and the G�/G� ratio were negatively correlated with the td

values, as were total PLFAs, bacterial PLFAs, G� bacterial PLFAs,G� bacterial PLFAs, actinomycetous PLFAs, and fungal PLFAs.

Responses of the soil microbial community to E. coli O157:H7. The results showed that the concentrations of most individualPLFAs were decreased after incubation with E. coli O157:H7 for 25days, except for the 15:00 and cy19:0 PLFAs (data not shown). Theconcentrations of total PLFAs, bacterial PLFAs, and G� bacterialPLFAs were also slightly lower in test soils (S0 to S15) with E. coliO157:H7 after 25 days of incubation than in soils without E. coliO157:H7 (Fig. 1). The levels of fungal PLFAs in soils S0, S7, andS15 declined significantly (P, �0.05). Moreover, actinomycetousPLFA levels also declined significantly (P, �0.05) in the test soils,except for S0. In contrast, G� bacterial PLFA levels increased

slightly after incubation with E. coli O157:H7 for 25 days. Theratios of the particular PLFAs also changed after 25 days of incu-bation (Fig. 2). The G�/G� ratio declined after E. coli O157:H7was inoculated into the soils. The B/F ratio in the test soils in-creased significantly after incubation with E. coli O157:H7 for 25days (P, �0.05). Meanwhile, the ratio of normal saturated PLFAsto monounsaturated PLFAs (S/M) also increased slightly. How-

FIG 2 Ratios of particular PLFAs in test soils. Data are means � 1 standard deviation for three replicates. Shown are the ratios of bacterial to fungal PLFAs (B/F),of G� to G� bacterial PLFAs (G�/G�), of cyclopropyl PLFAs to their precursors (cy/pre), and of saturated to monounsaturated PLFAs (S/M). C, W, and E areexplained in the legend to Fig. 1. The same lowercase letters indicate no significant differences, and different lowercase letters indicate significant differences (P �0.05) among C, W, and E. The soil codes (S0, S1, S4, S7 and S15) are explained in the footnote to Table 1.

FIG 3 Redundancy analysis (RDA) of the PLFA data set for fresh soils, usingsoil microbial composition and the survival time of E. coli O157:H7 (td) in thetest soils. B, bacterial PLFAs; G�, Gram-negative bacterial PLFAs; G�, Gram-positive bacterial PLFAs; A, actinomycetous PLFAs; F, fungal PLFAs; B/F, ratioof bacterial to fungal PLFAs; G�/G�, ratio of G� to G� bacterial PLFAs.

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ever, the untreated soils and those incubated with E. coli O157:H7exhibited various differences in the ratio of cyclopropyl fattyacids to their precursors (cy/pre ratio). The cy/pre ratios of S0,S1, and S4 increased after E. coli O157:H7 inoculation. How-ever, the trends in S7 and S15 were opposite those for S0, S1,and S4.

Figure 4 shows the effects of the invading E. coli O157:H7 onthe soil microbial community by principal component analysis(PCA). The first principal component (PC1, accounting for 60.0%of the variation) and the second principal component (PC2, ac-counting for 20.3% of the variation) together explained 80.3% ofthe variance in the PLFA data. Moreover, the total PLFAs andsignature microbial groups were most closely reflected in PC1,whereas PC2 explained mainly the selected PLFA ratios (Fig. 4A).The invasion of E. coli O157:H7 showed some effects on the soilmicrobial community. After incubation with E. coli O157:H7 for25 days, the PC2 loading values increased while the PC1 loadingvalues decreased (Fig. 4B).

DISCUSSION

The results of this study showed negative correlations between theE. coli O157:H7 survival time (td) and 14 PLFAs (Table 2) andbetween td and the G�/G� ratio (Fig. 3), which agreed with pre-vious findings that the indigenous microorganisms had a negativeeffect on E. coli O157:H7 survival due to predation, substrate com-petition, and antagonism (11, 15, 19). The G�/G� ratio can be anindicator of the structure of soil bacterial communities, suggestingthat the structure of the bacterial communities affects the survivalof E. coli O157:H7. In bacterial communities, bacteria can pro-duce bacteriocins, which can inhibit and even kill other strains orspecies with a similar niche or limit the advance of neighboringcells (36). Furthermore, bacteriocin production may be more im-portant in the invasion of niches than in obtaining nutrients (37).It has long been known that diverse pathogens can be suppressedin some agricultural soils (19, 38). Previous studies reported thatsome G� bacteria, such as Bacteroidetes, Gammaproteobacteria,Firmicutes, and Proteobacteria, were already known as environ-mentally stable populations of E. coli O157:H7-suppressing bac-teria (19, 38). In contrast to G� bacteria, G� bacteria such asActinobacteria and Listeria welshimeri correlated positively with E.coli O157:H7 survival (19, 39). Hence, we suppose that G� bacte-ria may produce bacteriocins that suppress E. coli O157:H7 to agreater extent than those of G� bacteria. In other words, thegreater antagonism of G� bacteria than of G� bacteria may ex-plain the significant negative correlation between the G�/G� ratioand td. In addition, microbial suppression may be harnessed todevelop new options for mitigating the risk and limiting the dis-persal of zoonotic bacterial pathogens in the environment (38).Therefore, the antagonism between E. coli O157:H7 and specificgroups of microorganisms may also have great potential for thebioremediation of E. coli O157:H7-contaminated soil.

Following inoculation, microorganisms undergo a variety ofprocesses, including growth, death, physiological adaption, genetransfer, and interactions with the indigenous microbial commu-nity (20, 40). Irrespective of plastic-greenhouse cultivation peri-ods, changes in the soil microbial community caused by the in-vading E. coli O157:H7 were detected in this study (Fig. 1, 2, and4B). Levels of total PLFAs, bacterial PLFAs, and G� bacterialPLFAs in the test soil declined slightly after 25 days of incubationwith E. coli O157:H7 (Fig. 1). Meanwhile, actinomycetous PLFAlevels declined significantly (P, �0.05) in this study (Fig. 1). Acti-nomycetes are widely distributed in soils. With variable physio-logical and metabolic properties, they can decompose and miner-alize naturally occurring compounds such as cellulose and chitin,thus playing a crucial role in organic-matter turnover (19). Inter-estingly, fungal PLFAs were also observed to decline significantlyafter invasion by E. coli O157:H7 (Fig. 1). Fungal growth is carbonlimited. Hence, this decline was probably linked to the decrease inthe levels of actinomycetes and G� bacteria. In this study, levels ofactinomycetes and fungi declined markedly as a result of E. coliO157:H7 invasion, suggesting that this pathogen may affect or-ganic-matter turnover due to the decline of the functional micro-bial community. However, G� bacterial PLFAs showed a slightincrease after incubation with E. coli O157:H7 for 25 days (Fig. 1).This also revealed that E. coli O157:H7 had greater antagonisticeffects on G� bacteria than on G� bacteria. Furthermore, someG� bacteria can form spores and exist in environments with loworganic carbon levels (41). An increase in the number of G� bac-

FIG 4 Principal component analysis (PCA) results for the PLFA data set(A) and the test soil samples (B). The abbreviations for the PLFA data setare explained in the legend to Fig. 2. C, W, and E are explained in the legendto Fig. 1. The soil codes (S0, S1, S4, S7, and S15) are explained in thefootnote to Table 1.

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teria may indicate a progressive change from copiotrophic tomore oligotrophic conditions (30). This implies that a morestressful soil environment was induced by the changed soil micro-bial community as a result of E. coli O157:H7 inoculation into thesoil.

In addition, ratios of the particular PLFAs are considered indi-cators of environmental stress (4, 31). As the levels of G� and G�

bacterial PLFAs decreased or increased after incubation with E.coli O157:H7 for 25 days (Fig. 1), the ratio of G� bacterial PLFAsto G� bacterial PLFAs (G�/G�) declined slightly in this study(Fig. 2). The ratio of normal saturated PLFAs to monounsaturatedPLFAs (S/M), which is considered very important for evaluatingcommunities and environmental conditions (4), can be used toindicate substrate bioavailability in the soil environment (31). Alower S/M ratio may be attributed to a higher rate of nutrientturnover (2, 42). In this study, the S/M ratio increased after 25days of incubation with E. coli O157:H7 (Fig. 2). This probablyimplied that the nutrient turnover ability in soils had declinedafter E. coli O157:H7 inoculation. Further, the B/F ratio was in-creased significantly (P � 0.05) after 25 days of incubation with E.coli O157:H7 (Fig. 2). Generally, lower B/F ratios are suggested toindicate a more sustainable ecosystem, with lower impact on theenvironment (43). Therefore, we speculated that a decrease in thesustainability of the ecosystem may be attributed to the inocula-tion of E. coli O157:H7. Moreover, the proportion of cyclopropylfatty acids, especially the cy/pre ratio, could also be affected by thenutrient status and physiological state of the microbial commu-nity (44). A higher cy/pre ratio indicated that the microbial com-munities were suffering greater environmental disturbance (45).The trend of the cy/pre ratio in the test soils differed with thedifferent plastic-greenhouse cultivation periods. The cy/pre ratiosin soils with shorter plastic-greenhouse cultivation periods (S0,S1, and S4) increased after incubation with E. coli O157:H7 for 25days but decreased in soils with longer plastic-greenhouse cultiva-tion periods (S7 and S15) (Fig. 2). This implies that the response ofindigenous microorganisms in the soil to E. coli O157:H7 invasionwas also related to the plastic-greenhouse cultivation periods.

As shown in Table 1 and Fig. 1, 2, and 4B, soil microbial com-munities were also influenced by plastic-greenhouse cultivationperiods. For instance, the contents of total PLFAs and specificmicrobial groups were higher in the open-field soil (S0) than inthe plastic-greenhouse soils (S1 to S15) (Fig. 1). However, somechanges in the soil microbial community in test soils were ob-served after 25 days of incubation with E. coli O157:H7 (Fig. 1, 2,and 4B). As shown in Fig. 4B, microbial communities in soilsincubated with E. coli O157:H7 for 25 days differed from those infresh soils and in soils incubated without E. coli O157:H7 for 25days. The changes in PC1 and PC2 loading values for the soilsamples incubated with E. coli O157:H7 were consistent with theresults in Fig. 1 and 2. Namely, the levels of total PLFAs and sig-nature microbial groups were decreased after E. coli O157:H7 in-oculation, while the B/F and S/M ratios increased.

Conclusions. The interaction of E. coli O157:H7 and the mi-crobial community in soils from vegetable fields were studied us-ing the PLFA method. The results showed that G� bacteria sup-press the survival of E. coli O157:H7 to a great extent. The invasionof E. coli O157:H7 changed the soil microbial community to someextent, making the environment stressful and unstable. In soilsincubated with E. coli O157:H7 for 25 days, the contents of acti-nomycetous PLFAs and fungal PLFAs were significantly decreased

(P, �0.05), while the S/M ratio and the B/F ratio were increased,over those in soils without E. coli O157:H7 after 25 days of incu-bation. This indicates that the toxicity of E. coli O157:H7 presentsnot only in its pathogenicity but also in its effect on soil micro-ecology, which deserves more attention than it has received todate. Hence, it is critical to control the dissemination of E. coliO157:H7 into the open environment in order to reduce its nega-tive environmental impacts and ensure food safety and publichealth.

ACKNOWLEDGMENTS

This work was supported by the Major Program of the National NaturalScience Foundation of China (41130532) and by grant 40971255 from theNational Natural Science Foundation of China.

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