RESEARCH ARTICLE Influence of bacterial communities based on 454-pyrosequencing on the survival of Escherichia coli O157:H7 in soils Jincai Ma1,3, Abasiofiok M. Ibekwe1, Ching-Hong Yang2 & David E. Crowley3 1USDA-ARS U. S. Salinity Laboratory, Riverside, CA, USA; 2Department of Biological Sciences, University of Wisconsin, Milwaukee, WI, USA; and 3Department of Environmental Sciences, University of California, Riverside, CA, USA Correspondence: Abasiofiok M. Ibekwe, Abstract USDA-ARS-U. S. Salinity Laboratory, 450 W. Big Springs Rd, Riverside, CA 92507, Shiga toxin-producing Escherichia coli O157:H7 has been implicated in many USA. Tel.: +1 951 369 4828; foodborne illnesses. In this study, survival of E. coli O157:H7 in 32 soils from fax: +1 951 342 4964; California (CA) and Arizona (AZ) was investigated. Our goal was to correlate e-mail: [email protected] the survival time of E. coli O157:H7 in soils with 16S rRNA pyrosequencing based bacterial community composition. Kohonen self-organizing map of sur- Received 28 September 2012; revised 14 vival and associated soil chemical, physical and biological variables using artifi- January 2013; accepted 23 January 2013. Final version published online 26 February cial neural network analysis showed that survival of E. coli O157:H7 in soils 2013. was negatively correlated with salinity (EC), but positively correlated with total nitrogen (TN) and water soluble organic carbon (WSOC). Bacterial diversity as DOI: 10.1111/1574-6941.12083 determined by the Shannon diversity index had no significant (P = 0.635) effect on ttd, but individual bacterial phyla had different effects. The survival Editor: Julian Marchesi of E. coli O157:H7 was positively correlated with the abundances of Actinobac- teria (P < 0.001) and Acidobacteria (P < 0.05), and negatively correlated with Keywords those of Proteobacteria and Bacteroidetes (P < 0.05). Our data showed that spe- contamination; fresh produce; organic; cific groups of bacteria correlate with the persistence of E. coli O157:H7 in soils conventional. thus opening new ways to study the influence of certain bacterial phyla on per- sistence of this pathogen and other related pathogens in complex environ- ments. (2008) showed that the changes in microbial diversities Introduction and community compositions coincided with an enhance- Numerous studies have been carried out to investigate ment of the survival rate of the invading pathogen. The the fate of Escherichia coli O157:H7 in different agricul- basic explanation presented by van Elsas et al. (2011) was tural systems with major focus on the survival of E. coli that lowering of the complexity of the soil microbiota O157:H7 in soils and manure-amended soils (Jiang et al., probably resulted in a reduction in functional redun- 2002; Franz et al., 2008; Semenov et al., 2008). One of dancy, which enhances the chances for the introduced the most recent review articles (van Elsas et al., 2011) on microorganisms to occupy a niche in the system and per- survival of E. coli O157:H7 showed that temperature, soil sist as a member of the microbial community. They con- structure, and microbial communities were the most cluded that some organisms could be direct competitors important factors affecting survival (van Elsas et al., by occupying the same niche, whereas others might have 2011). These authors showed from their previous studies antagonistic or predatory activities. However, these (van Elsas et al., 2007) that the survival of E. coli O157: authors lamented the lack of data on impact of different H7 was inversely proportional to the diversity of the functional bacterial groups on survival of E. coli O157:H7. MICROBIOLOGY ECOLOGY MICROBIOLOGY microbial community established through differential Depending on the soil physical, chemical, and biologi- fumigation and regrowth activities. Using polymerase cal characteristics and environmental factors (e.g. temper- chain reaction–denaturing gradient gel electrophoresis ature and precipitation), the survival time of E. coli (PCR-DGGE), Franz et al. (2008) and Semenov et al. O157:H7 in soils varies from 1 week to 6 months, and ª 2013 Federation of European Microbiological Societies FEMS Microbiol Ecol 84 (2013) 542–554 Published by Blackwell Publishing Ltd. All rights reserved Survival of Escherichia coli O157:H7 in soils 543 even longer in some extreme cases (Maule, 2000; Mubiru Each sample (0–15 cm) was a composite of several indi- et al., 2000; Ibekwe et al., 2007, 2010; Ibekwe & Ma, vidual soil cores taken at 5-m intervals. Equal numbers of 2011; Ma et al., 2011). It would be interesting to investi- samples from conventional farming practice and organic gate the role of specific environmental variables, particu- farming practice were collected from each area (32 soils, larly functional bacterial groups, on the overall survival of 16 organically, 16 conventionally managed soils). Yuma E. coli O157:H7 using traditional microbial ecology and Imperial Valley shared similar weather conditions, approaches (e.g. PCR-DGGE), next-generation sequencing including MAT and MAP, due to the closeness of the two techniques (e.g. 454-pyrosequencing and Illumina), and locations in comparison with Salinas Valley, CA. Soil pH microarray protocol (e.g. PhyloChip). The low sequencing was between 6.7 and 8.0. Bagged samples were taken to depth of the traditional PCR cloning approach when the laboratory under ice. Vegetation, roots, and stones compared with the vast genetic diversity present in soils were removed and the soil sieved (< 2 mm). Soil physi- hindered a comprehensive characterization of the micro- cal, chemical, and some biological characteristics are as bial community structure. In this aspect, the current reported (Ma et al., 2012a, b). Total organic carbon (OC, community analyses typically represent a mere snapshot of %) and total nitrogen (TN, %) were determined using the dominant members, with little information on taxa with Flash 2000 NC Analyzers (Thermo Scientific, Lakewood, À medium to low abundances. High-throughput sequencing NJ). Soil microbial biomass carbon (MBC, mg kg 1) was is a promising method, as it provides enough sequencing extracted by the chloroform-fumigation–extraction depth to cover the complex microbial communities method (Vance et al., 1987). Salinity measured as electri- À (Shendure & Ji, 2008). For this reasons, we believe that cal conductivity (EC, dS m 1) of each soil was obtained the correlation of different bacterial phyla based on deep by determining the conductivity of soil water extract sequencing may provide some insights into some of the (water to soil ratio, 1 : 1, vol:wt) using a conductivity bacterial groups that may affect the persistence of E. coli meter (Oakton conductivity meter, Oakton, Houston, O157:H7 in soil. TX). In this study, the survival of Shiga toxin–producing E. coli O157:H7 strain EDL933 in 32 soils from three Bacterial strains major leafy green-producing areas of CA and AZ was investigated. The majority of E. coli O157:H7 outbreaks An E. coli O157:H7 strain EDL933 was used in this study have been associated with Salinas Valley area in CA, (ATCC 43895), as described previously (Ma et al., 2011). where most of the summer produce is grown compared Escherichia coli O157:H7 wild type was tagged with nali- toYuma,AZ,andImperialValley,CA,wherewinterproduce dixic acid in addition to rifampicin resistance to facilitate is grown. Our work expands on the above studies using the enumeration, and its growth curve in LB (Luria- 454-pyrosequencing-based technology to address the role Bertani) broth and survival in soils were found to be of different bacterial phyla on the survival of this patho- identical to those of the nontagged wild-type strain (Ma gen. In the current study, the survival data were analyzed et al., 2011). using the Weibull model (Mafart et al., 2002; Franz et al., 2008) and artificial neural network analysis using Kohonen Escherichia coli O157:H7 EDL933 survival in soils self-organizing map of E. coli survival, and the data were correlated with soil bacterial community composition and Cells were pregrown overnight in LB medium, harvested soil properties. by centrifugation (12 000 g) for 10 min, and washed with phosphate buffer (10 mM, pH 7.2) three times. The cell pellets were finally resuspended in sterile deionized water Materials and methods and used as inoculum. The cells were added in soils to a final density of about 0.5 9 107 CFU per gram soil dry À Soil collection and characterization weight (gdw 1) according to a method slightly adapted Soil samples were collected from three major fresh from Franz et al. (2008). About 500 g of the inoculated produce-growing areas: Salinas Valley area CA, Imperial soil was transferred to a plastic bag that was closed but, Valley, southern CA, and Yuma Valley, AZ. The sampling which had some holes at the top to allow air exchange as Global Positioning System (GPS) coordinates, mean previous described (Ma et al., 2011). The same amount annual temperature (MAT), and mean annual precipita- of noninoculated soil was also placed into another plastic tion (MAP), as well as other environmental parameters of bag, which was used as a noninoculated control, with sampling sites, were previously described (Ma et al., deionized water being added instead of cell suspension in 2012a, b; Table S1). The major fresh produce growing on triplicate plastic bags. The plastic bags were weighed and these locations during sampling were lettuce and spinach. incubated at 22 °C. Moisture content of the soil sample FEMS Microbiol Ecol 84 (2013) 542–554 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 544 J. Ma et al. was adjusted to 50% of WHC, and water concentration linear curve is observed. The scale parameter, d, repre- was maintained constantly during the course of an exper- sents the time (day) needed for first decimal reduction.