New Insights into the Formation of Viable but Nonculturable Escherichia coli O157:H7 Induced by High-Pressure CO 2

ABSTRACT The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO 2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l -serine and l -threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO 2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO 2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation. IMPORTANCE Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO 2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment.

Behavior of an Aeromonas hydrophila aroA Live Vaccine in Water Microcosms

ABSTRACT Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized. We previously constructed and characterized an aroA mutant of Aeromonas hydrophila and studied the protection afforded by this mutant as a live vaccine in rainbow trout. In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks. The aroA mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water. When the aroA strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain A. hydrophila AG2; after long periods of incubation, aroA cells were able to enter a viable but nonculturable state. Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity. Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two A. hydrophila strains at low temperatures (6 and 16°C). These results contribute to a better understanding of the behavior of the aroA strain in natural environments and suggest that the inactivation of the aroA gene may be beneficial for the safety of this live vaccine for aquacultures.

Identification of a low-threshold T-type calcium channel in bovine ciliary epithelial cells

The characteristics of an inward current in single pigmented cells from the bovine ciliary body were examined using the whole cell recording technique. The inward current appeared to activate at potentials 30 mV positive to a holding potential of -80 mV, although activation studies revealed that from more negative holding potentials the current activated at about -75 mV, peaked at around -10 mV, and reversed at a potential of +40 mV. The current was small, peak value -72 +/- 24 pA (n = 13), and exhibited rapid activation and slower inactivation kinetics, both processes being voltage sensitive. Inactivation occurred at holding potentials more positive than -120 mV and was complete at -50 mV with a half-maximal membrane potential of -88 mV. The overlap of the inactivation and activation curves between -75 and -50 mV means that, in the steady state, entry of calcium will be greatest in this range and calcium will thus enter the cell at the resting membrane potential. The current was resistant to tetrodotoxin and nifedipine. It was partially blocked by Cd2+ and almost completely blocked by Ni2+. It is concluded that this current is a low-threshold T-type calcium current.

International Migration, International Relations and Foreign Policy

Recent literature on migration, international relations and foreign policy is reviewed in this article, stressing applications of global systems paradigms, studies of state entry and exit rules, and anatomies of domestic policy-setting processes on migration. After a concise assessment of the contemporary theory of global political economy, the paper argues for seeking midrange generalizations on the international relations of migration. It also suggests that analysis begin with the policy-setting processes of the state. Especially through the use of comparative perspectives available from domestic policymaking studies and from the field of international comparative public policy, this approach offers the opportunity to fix empirically the political roles of transnational social forces, which often present themselves as participants in domestic policy contests. Promising future directions in the study of state-to-state relations are also evaluated, with the anticipation that verifying regional or other intermediate patterns of world migration politics may contribute to more general theories of international political economy.