Biology and Control of Human Pathogens on Fresh Produce
Location: Produce Safety and Microbiology Research
Title: Functional metagenomics of Escherichia coli O157:H7 interactions with spinach indigenous microorganisms during biofilm formation
Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 30, 2012
Publication Date: September 5, 2012
Citation: Carter, M.Q., Xue, K., Brandl, M., Liu, F., Wu, L., Louie, J.W., Mandrell, R.E., Zhou, J. 2012. Functional metagenomics of Escherichia coli O157:H7 interactions with spinach indigenous microorganisms during biofilm formation. PLoS One. 9:e44186.
Interpretive Summary: Escherichia coli O157:H7 naturally resides in ruminants, primarily cows, and is transmitted to humans mainly through food vesicles. Recent increases in produce-associated E. coli O157:H7 infections world-wide suggest that this enteric pathogen may have considerable survival fitness on fresh vegetables or fruits. In this study, we examined the growth of, and the biofilm formation by, E. coli O157:H7 on stainless steel surfaces in E. coli O157:H7-inoculated spinach juice. We found that at the early stage of biofilm formation, the indigenous microorganisms living on spinach leaves stimulated the surface attachment of the pathogen E. coli O157:H7. However, at the later stage of biofilm formation, spinach-associated microorganisms inhibited the E. coli O157:H7 population significantly. To gain a better understanding of the mechanisms underlying the interactions between the E. coli O157:H7 and the native microorganisms on spinach leaves, we applied the GeoChip-based metagenomic approach to investigate the diversity of the biofilm community derived from spinach leaf and the effect of E. coli O157:H7 on the community structure over time. We found that both the gene richness and the biofilm community dissimilarity decreased significantly in the E. coli O157:H7 inoculated biofilm compared with the corresponding control biofilm (without E. coli O157:H7). Metagenomics revealed that the abundances of many genes involved in cycling carbon, nitrogen and phosphorus decreased significantly in pathogen-inoculated biofilms. We therefore conclude that the metabolic potential of E. coli O157:H7 in utilizing spinach nutrients contributes greatly to its survival fitness on spinach. Competition for essential macronutrients is likely the primary interaction between the E. coli O157:H7 and the indigenous spinach-biofilm species.
The increase in foodborne outbreaks worldwide attributed to fresh fruit and vegetables suggests that produce may serve as an ecological niche for enteric pathogens. Here we examined the interaction of E. coli O157:H7 (EcO157) with spinach leaf microflora during co-colonization and establishment of a mixed biofilm on a stainless steel surface. We observed a positive effect of spinach-associated microbes on the initial attachment of EcO157, but an antagonistic effect on the EcO157 population at the later stage of biofilm formation. Metagenomic analyses of the spinach biofilm community with the GeoChip revealed an extremely diverse community represented by ~ 23,000 DNA probes primarily from 11 bacterial- and two fungal phyla. Presence of EcO157 in the mixed biofilm resulted in a significant decrease in the community a-diversity (t test, P<0.05), indicating a putative competition between the pathogen and indigenous spinach microbiota. The decrease in the ß-diversity of the EcO157-inoculated biofilm at 48 h (ANOVA, P<0.05) suggested a convergent shift in functional composition in response to EcO157 invasion. The success of EcO157 in the mixed biofilm is likely associated with its metabolic potential in utilizing spinach nutrients: the generation time of EcO157 in spinach lysates at 28°C is ~ 38 min, which is comparable to that in rich broth. The significant decrease in the abundance of many genes involved in carbon, nitrogen, and phosphorus cycling in the EcO157-inoculated biofilms (t test, P<0.05) support the hypothesis that competition for essential macronutrients is the primary interaction between the EcO157 and indigenous spinach-biofilm species.