Survival and recovery of Escherichia coli O157:H7 during starvation at sub-optimal temperatures

Escherichia coli O157:H7 is an important foodborne pathogen with a dual lifestyle of growth within a host and persistence in non-host environments. Environmental survival of E. coli O157:H7 is particularly important in New Zealand due to the increasing trend in notified clinical cases, with most being associated with rural environments rather than contaminated regulated foods. Starvation and a decrease in temperature are characteristic of the conditions encountered by bacteria during transition from a reservoir host, normally a ruminant, to the environment; cf. feast to famine. These conditions are known to induce bacterial entrance into the viable but non-culturable (VBNC) state where cell viability can only be detected using culture-independent methods with the number of colonies recovered decreasing over the duration of the starvation period. Findings from this study showed that formation of a VBNC sub-population can be induced by starving E. coli O157:H7 at sub-optimal temperatures. Five E. coli O157:H7 strains were used in this study; NCTC12900 as a reference strain, three New Zealand clinical isolates and a New Zealand bovine isolate. Cells were starved in phosphate buffered saline for 84 days at sub-optimal temperatures. Cell viability was determined using the SYTO 9-PI staining system coupled with a haemocytometer counting method developed in this study. Cell culturability was assessed using the nutrient-rich tryptic soy agar (TSA), nutrient-limited plate count agar (PCA), and PCA with additional supplements. The distribution of colony size provided information on the population dynamics of starved cells and their ability to recover on agar. The role of quorum sensing (LuxS/autoinducer-2) on starved cell recovery was investigated by supplementing PCA with quorum sensing molecules and using a mutant strain deficient in luxS, a gene encoding the synthase of autoinducer-2. The ability of starved and non-starved cells to metabolise single carbon sources were compared using the Biolog MicroPlatesTM. When held at 4°C, a three- to six-log reduction in culturability on PCA was observed after 84 days of starvation. The reduction in culturability observed for cells starved at 15°C was between one to two logs on PCA. Despite the decline in culturability on PCA, the number of viable cells as determine by LIVE/DEAD® BacLightTM staining remained within one log of the initial viable count. Supplementation of PCA with 0.2% sodium pyruvate resulted in a significant increase in the number of colonies recovered from 4°C-starved cultures for all E. coli O157:H7 strains tested. Recovery on TSA was consistently at least two-logs higher than recovery on PCA for E. coli O157:H7 cells after 84 days of starvation at 4°C. The distribution of colony diameter was markedly wider for 4°C-starved cells compared to non-starved cells when recovered under aerobic conditions. In addition to the strain variation observed, culturability of starved E. coli O157:H7 was found to be dependent on the starvation temperature, the nutrient level of recovery media, and the presence of specific resuscitation factors such as pyruvate. While the culturability phenotype was similar between reference, clinical and bovine E. coli O157:H7 strains, metabolic profiles determined using carbon source MicroPlatesTM differed. Analysis of the carbon utilisation kinetics allowed comparison of carbon metabolism between exponential phase, stationary phase and starved E. coli O157:H7. The duration of lag in substrate utilisation was significantly different between starved and non-starved cells. Supplementing PCA with quorum sensing molecules and using a mutant strain deficient in luxS, a gene encoding the synthase of autoinducer-2, did not result in a significant improvement in the recovery of starved VBNC cells. Based on current knowledge, models for the change in E. coli O157:H7 population dynamics during its lifecycle, the role of pyruvate resuscitation of VBNC cells, and factors involved in E. coli O157:H7 infection of bovine and human hosts were proposed. The ability of New Zealand clinical and bovine isolates to enter the VBNC state, which was demonstrated in this study, highlighted the potential for culture-based assessment methods to underestimate the number of viable E. coli O157:H7. While reports of the use of pyruvate for resuscitating VBNC cells are in the literature, this study is the first to propose a mechanism of pyruvate resuscitation. By understanding the physiology of VBNC cells and mechanism of their resuscitation, it may be possible to eliminate the potential for underestimation of viable E. coli O157:H7 in the environment and in food by tailoring culture methods to promote their growth