Relationships between Escherichia coli cells and the surrounding medium during survival processes

In Escherichia coli, during survival under adverse conditions, namely starvation and luminous radiation, two things occur. On the one hand organic substances are released into the surrounding medium and on the other there is a transition from the culturable state to viable but non-culturable (VBNC). An analysis of organic molecules released into the surrounding medium showed the presence of proteins, dissolved free amino acids, and dissolved monomeric carbohydrates. The concentration of these substances in the medium changed with exposure time, type of stress and type of molecule. The proteins accumulated in the medium and in some cases their identification revealed the presence of components of the outer membrane. Variations in the concentration of amino acids and carbohydrates point to a twofold process of excretion and uptake. Indeed, cell free supernatants supported the growth of several generations of a population of 10(4) cells ml(-1). The survival of E. coli in supernatants previously colonized by cells in the VBNC state was greater than that observed in the control experiments, with a short delay in the loss of culturability. It was thus clear that organic molecules released into the medium play a role in the transition from culturable to VBNC state.     

Cell wall chemical composition of Enterococcus faecalis in the viable but nonculturable state

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.     

Proteomic characterization of enterohemorrhagic escherichia coli O157:H7 in the oxidation-induced viable but non-culturable state

Enterohemorrhagic Escherichia coli (EHEC) O157 strain F2, a food isolate of an outbreak, is resistant to oxidative stress, but has increased stress-sensitivity after passage through mice. The stress-sensitive variant of F2 (designated MP37) has decreased culturability, but retains membrane integrity under stress conditions, indicating that the cells enter a viable but non-culturable (VBNC) state. Proteomic analyses revealed that MP37 in the VBNC state had decreased levels of some oxidation-responsive factors (AhpCF, AceF), but it markedly increased levels of outer membrane protein W (OmpW). Because F2 expressed higher levels of some ribosome-associated proteins (RaiA, S6, Bcp) than MP37, the effect of animal passage on the induction of the VBNC state in the EHEC O157 cells might be due to ribosomal activity.     

Inability of Escherichia coli to resuscitate from the viable but nonculturable state

After induction of the viable but nonculturable (VBNC) state in Escherichia coli populations, we analysed abiotic and biotic factors suggested to promote the resuscitation process. The response to the stressing conditions implied the formation of three subpopulations, culturable, VBNC and nonviable. In most adverse situations studied, the VBNC subpopulation did not represent the dominant fraction, decreasing with time. This suggests that, in most cases, the VBNC is not a successful phenotype. Combining methods of dilution and inhibition of remaining culturable cells, we designed a working protocol in order to distinguish unequivocally between regrowth and resuscitation. Reversion of abiotic factors inducing nonculturability as well as prevention of additional oxidative stress did not provoke resuscitation. Participation of biotic factors was studied by addition of supernatants from different origin without positive results. These results indicate that the E. coli strain used is not able to resuscitate from the VBNC state. VBNC cells release into the surrounding medium, and could thus aid in the survival of persisting culturable cells. The formation of a VBNC subpopulation could thus be considered as an adaptive process, designed for the benefit of the population as a whole.     

Cell Wall Chemical Composition of Enterococcus faecalis in the Viable but Nonculturable State

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.     

细菌在逆境条件下膜脂肪酸变化的研究进展

细胞膜是控制细菌细胞进行物质交换的屏障。在逆境条件下,细菌通过改变细胞膜脂肪酸的组分和含量,以调整适当的膜流动性和适应性,保护细胞膜免受不利和多变逆境条件的影响。有些细菌在逆境胁迫的条件下会进入活的但不可培养的(Viable but non-culturable, VBNC)状态。总结了细菌几种逆境胁迫及其诱导因子,并论述了细菌和部分具有VBNC态细菌在逆境胁迫下膜脂肪酸的种类及含量的变化、以及脂肪酸检测方法的研究进展,为进一步解析细菌逆境胁迫机制提供参考。     

Systematic identification of the subproteome of Escherichia coli cell envelope reveals the interaction network of membrane proteins and membrane-associated peripheral proteins

Membrane proteins of Gram-negative bacteria are key molecules that interface the cells with the environment. Despite recent proteomic identification of numerous oligomer proteins in the Escherichia coli cell envelope, the protein complex of E. coli membrane proteins and their peripherally associated proteins remain ill-defined. In the current study, we systematically analyze the subproteome of E. coli cell envelope enriched in sarcosine-insoluble fraction (SIF) and sarcosine-soluble fraction (SSF) by using proteomic methodologies. One hundred and four proteins out of 184 spots on 2D electrophoresis gels are identified, which includes 31 outer membrane proteins (OMPs). Importantly, our further proteomic studies reveal a number of previously unrecognized membrane-interacting protein complexes, such as the complex consisting of OmpW and fumarate reductase. This established complete proteomic profile of E. coli envelope also sheds new insight into the function(s) of E. coli outer envelope.     

Differential effects of temperature and starvation on induction of the viable-but-nonculturable state in the coral pathogens Vibrio shiloi and Vibrio tasmaniensis

We compared induction of the viable-but-nonculturable (VBNC) state in two Vibrio spp. isolated from diseased corals by starving the cells and maintaining them in artificial seawater at 4 and 20 degrees C. In Vibrio tasmaniensis, isolated from a gorgonian octocoral growing in cool temperate water (7 to 17 degrees C), the VBNC state was not induced by incubation at 4 degrees C after 157 days. By contrast, Vibrio shiloi, isolated from a coral in warmer water (16 to 30 degrees C), was induced into the VBNC state by incubation at 4 degrees C after 126 days. This result is consistent with reports of low-temperature induction in several Vibrio spp. A large proportion of the V. tasmaniensis population became VBNC after incubation for 157 days at 20 degrees C, and V. shiloi became VBNC after incubation for 126 days at 20 degrees C. Resuscitation of V. shiloi cells from cultures at both temperatures was achieved by nutrient addition, suggesting that starvation plays a major role in inducing the VBNC state. Our results suggest that viable V. shiloi could successfully persist in the VBNC state in seawater for significant periods at the lower temperatures that may be experienced in winter conditions, which may have an effect on the seasonal incidence of coral bleaching. For both species, electron microscopy revealed that prolonged starvation resulted in transformation of the cells from rods to cocci, together with profuse blebbing, production of a polymer-like substance, and increased membrane roughness. V. shiloi cells developed an increased periplasmic space and membrane curling; these features were absent in V. tasmaniensis.     

Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state

When exposed to stress-provoking environmental conditions such as those of ground waters, many medically important bacteria have been shown to be capable of activating a survival strategy known as the viable but non-culturable (VBNC) state. In this state bacteria are no longer culturable on conventional growth media, but the cells maintain their viability and pathogenicity genes/factors and can start dividing again, in a part of the cell population, upon restoration of favourable environmental conditions. Little is known about the genetic mechanisms underlying the VBNC state. In this study we show evidence of involvement of the rpoS gene in persistence of Escherichia coli in the VBNC state. The kinetics of entry into the non-culturable state and duration of cell viability were measured in two E. coli mutants carrying an inactivated rpoS gene and compared with those of the parents. For these experiments, laboratory microcosms consisting of an artificial oligotrophic medium incubated at 4 degrees C were used. The E. coli parental strains reached the non-culturable state in 33 days when the plate counts were evaluated on Luria-Bertani agar containing sodium pyruvate, whereas cells of the rpoS mutants lost their culturability in only 21 days. Upon reaching unculturability the parents yielded respiring cells and cells with intact membranes for at least the next three weeks and resuscitation was allowed during this time. In contrast, the RpoS- mutant cells demonstrated intact membranes for only two weeks and a very restricted (<7 days) resuscitation capability. Guanosine 3',5'-bispyrophosphate (ppGpp) acts as a positive regulator during the production and functioning of RpoS. A mutant deficient in ppGpp production behaved like the rpoS mutants, while overproducers of ppGpp displayed a vitality at least comparable to that of RpoS+ strains. These results suggest that the E. coli parental strains enter the VBNC state which lasts for, at least, three weeks, after which apparently all the cells die. The rpoS mutants do not activate this survival strategy and early die. This implies involvement of the rpoS gene in E. coli persistence in the VBNC state.     

Entry of Vibrio cincinnatiensis into viable but nonculturable state and its resuscitation

Aims:  The aim was to characterize the viable but nonculturable (VBNC) state of Vibrio cincinnatiensis and its resuscitation.
Methods and Results:  Vibrio cincinnatiensis VIB287 was cultured in sterilized seawater microcosms at 4°C. Plate counts, direct viable counts and total counts were used. A large population of the V. cincinnatiensis became nonculturable after approx. 50 day at 4°C. Electron microscopy revealed that the VBNC cells changed from rod to coccoid and decreased in size. Resuscitation of VBNC cells was achieved by temperature upshift in nutrition of yeast extract and peptone by addition of catalase or compound vitamin B. The VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately. No death was observed in the group inoculated with the VBNC cells.
Conclusions:  Vibrio cincinnatiensis VIB287 could enter VBNC state in adverse environments. Resuscitation of VBNC cells occurred by addition of compound vitamin B or catalase to VBNC cells containing nutrient. The resuscitative cells might retain their pathogenicity.
Significance and Impact of the Study:  The study confirmed that V. cincinnatiensis could enter into VBNC state in seawater at low temperature and resuscitated. The resuscitative cells retained their pathogenicity, which may be important in future studies of ecology of V. cincinnatiensis .     

Long-term survival and plasmid maintenance of Escherichia coli in marine microcosms

Abstract The survival pattern and plasmid maintenance of Escherichia coli was examined in an artificial seawater microcosm. It was found that the three strains of E. coli (EK3C, H10407 and 34309) included in the study were able to maintain a portion of cells in the culturable phase for at least 3 years in artificial seawater. Along with retaining culturability, that portion of the cell population also maintained their indigenous plasmids over the 3-year period. It is concluded that cells of E. coli maintaining culturability in seawater are selectively adapted to the salinity of seawater, remaining in a culturable state. The results of the study are significant in that it has been assumed by many public health authorities that E. coli cannot survive, without nutrient addition, in seawater for long periods of time, i.e., years of exposure to seawater.     

Induction of viable but nonculturable Escherichia coli O157:H7 in the phyllosphere of lettuce: a food safety risk factor

Escherichia coli O157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate of E. coli O157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction of E. coli O157:H7 Tn7gfp transformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), both E. coli O157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (10(9) and 10(6) E. coli O157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log(10) cells but did not detect culturable cells. These findings indicate that E. coli O157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.     

The survival response of Escherichia coli K12 in a natural environment

To verify the hypothesis of cryptic growth and viable but nonculturable (VBNC) state, survival responses of Escherichia coli cells were examined under oligotrophic microcosm conditions for an extended period. In the case of filtered distilled water at 4°C, E. coli cells definitely entered the VBNC state within 56 days. However, culturability and viability increased while the total number of cells declined after 110 days. This phenomenon can be explained by considering three possible states. The first is the existence of the VBNC state, the second is cryptic growth, and the third is the death of E. coli cells. In the case of artificial seawater at 4°C, VBNC E. coli cells confirmed the existence of two log units of elongated VBNC cells. Moreover, elongated VBNC cells showed the most significant change among all the other transformed cells. Also, E. coli cells in microcosms at 28°C indicated the entrance to the classical starvation survival state. In resuscitation tests, 1% diluted Luria-Bertani agar medium showed the highest level of resuscitation among amended agar media. To evaluate the survival ability of E. coli cells in the activated sludge samples, we used an E. colistrain XL-1 blue containing plasmids pQ2 including GFPcDNA (XL/GFP). In supernatant of activated sludge (SUP) at 28°C, XL/GFP cells entered the VBNC state after 10 days, whereas existence of VBNC cells was not detectable in resuspended activated sludge (ACT) at 28°C.     

Mode of action of gramicidin S on Escherichia coli membrane

The action of a cationic antibiotic gramicidin S on the outer and cytoplasmic membranes of Escherichia coli was studied. It was found that gramicidin S disrupted the permeability barrier of the outer membrane, permitting the permeation of an antibiotic ionophore, this being similar to the action of the dimer in compound 48/80 (Katsu, T., Shibata, M. and Fujita, Y. (1985) Biochim. Biophys. Acta 818, 61-66). However, differently from the dimer, gramicidin S further stimulated the efflux of K+ through the cytoplasmic membrane of E. coli. The time course of K+ permeability change accorded well with that of change in the viability of E. coli cells. These changes occurred at temperatures above the phase transition of the cytoplasmic membrane. This temperature range differed greatly from the case of polymyxin B, a polycationic antibiotic acting at temperatures above the phase transition of the outer membrane. We discuss the mode of gramicidin S action on the cytoplasmic membrane of E. coli, in comparison with the results on red blood cells and liposomes.     

Detection of Iss and Bor on the surface of Escherichia coli

AIMS: To confirm the presence of Iss and Bor on the outer membrane of Escherichia coli using Western blots of outer membrane protein (OMP) preparations and fluorescence microscopy, and explore the use of fluorescence microscopy for the detection of avian pathogenic E. coli (APEC) and diagnosis of avian colibacillosis. METHODS AND RESULTS: Knockout mutants of iss and bor were created using a one-step recombination of target genes with PCR-generated antibiotic resistance cassettes. Anti-Iss monoclonal antibodies (Mabs) that cross-react with Bor protein were used to study the mutants relative to the wild-type organism. These Mabs were used as reagents to study OMP preparations of the mutants with Western blotting and intact E. coli cells with fluorescence microscopy. Iss and Bor were detected in Western blots of OMP preparations of the wild type. Also, Iss was detected on Deltabor mutants, and Bor was detected on Deltaiss mutants. Iss and Bor were also detected on the surface of the intact, wild-type cells and mutants using fluorescence microscopy. CONCLUSIONS: These results demonstrate that Bor and Iss are exposed on E. coli's outer membrane where they may be recognized by the host's immune system. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report confirming Iss' location in the outer membrane of an E. coli isolate. Such surface exposure has implications for the use of these Mabs for APEC detection and colibacillosis control.     

Proteomic Analysis of Protein Expression in the Induction of the Viable But Nonculturable State of Vibrio harveyi SF1

Vibrio harveyi has been reported to enter into a viable but nonculturable (VBNC) state. One marine V. harveyi strain, SF1 became nonculturable when incubated in seawater microcosm at 4 °C within 60 days. We investigated protein expression in the exponential phase of V. harveyi SF1 and compared it to the VBNC state. Cytosolic proteins were resolved by two-dimensional polyacrylamide gel electrophoresis using pH 4–7 linear gradients. Among these proteins, sixteen proteins which were strongly downregulated or upregulated in the VBNC cells were identified by MALDI-TOF-TOF mass spectrometry. The results indicated that the differentially expressed proteins were mainly focused on stress response proteins and key components of central and intermediary metabolism, like carbohydrate metabolism, transport, and translation. This study provided clues for understanding the mechanism of adaptation to the VBNC state.     

Persistence of Enterococcus faecalis in aquatic environments via surface interactions with copepods

Several human pathogens and fecal-pollution indicators may persist as viable organisms in natural environments, owing to their ability to activate different types of survival strategies. These strategies include adhesion on both abiotic and biotic surfaces and the entrance to the so-called viable but nonculturable (VBNC) state. In an 18-month survey for the detection of enterococci in both lake water and seawater, C. Signoretto et al. (Appl. Environ. Microbiol. 70:6892-6896, 2004) have shown that Enterococcus faecalis was detected mostly bound to plankton and in the VBNC state. In the present study, we show that in vitro adhesion of E. faecalis to copepods accelerated the entry of cells into the VBNC state relative to that of planktonic bacteria. VBNC E. faecalis cells maintained their adhesive properties to copepods and chitin (the main component of the copepod carapace), though to a reduced extent in comparison with growing cells. Sugar competition experiments showed interference with adhesion to both copepods and chitin by GlcNAc and only to copepods by D-mannose. Four enterococcal cell wall proteins present in both growing and VBNC cells and lipoteichoic acid were shown to be capable of binding chitin. The results indicate that copepods may represent an additional environmental reservoir of enterococci, thus suggesting the advisability of redesigning the protocols currently used for microbial detection during the evaluation of the microbiological quality of environmental samples.     

Protein II influences ferrichrome-iron transport in Escherichia coli K12.

Ferrichrome-promoted iron uptake in Escherichia coli K12 is strictly dependent upon the tonA gene product, a 'minor' outer membrane protein. By selection for mutants of E. coli resistant to phages which require 'major' outer membrane proteins as receptors, strains with pronounced protein deficiencies were constructed. Such strains were tested for anomalous behaviour of ferrichrome transport. No significant differences in iron uptake were detected in E. coli K12 strains with markedly reduced amounts of protein I. However, a reduction in the initial velocity (up to 40%) was observed in E. coli deficient in outer membrane protein II. This difference was only evident when cells were grown under iron-starvation conditions; it was abolished when cells were grown in rich medium. Kinetic parameters for ferrichrome transport were determined for maximum velocity but for Km; double reciprocal plots showed a biphasic nature, probably attributable to a limited number of outer membrane binding sites and to the multi-component nature of the ferrichrome-iron transport system.     

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr).     

Ammonia-induced cell envelope injury in Escherichia coli and Enterobacter aerogenes

Ammonia-induced cell envelope injury was examined in pure cultures of Escherichia coli and Enterobacter aerogenes. Cell injury, as determined by the ratio of colony-forming units on m-T7 agar to colony-forming units on m-Endo agar, increased with exposure to increasing concentrations of ammonia. Cell envelopes appeared to be the site of injury as indicated by increasing susceptibility to lysozyme with increasing ammonia concentration. Cells exposed to ammonia also exhibited more cellular leakage than control cells. Leakage from cells exposed to ammonia included proteins, and all leaked substances increased in concentration as ammonia concentrations increased. The concentration of 2-keto-3-deoxyoctonate (KDO) in the outer membrane of E. coli increased with ammonia exposure, while KDO concentration in the outer membrane of E. aerogenes decreased. The results suggest that exposure of E. coli cells to high concentrations of ammonia disrupts the outer membrane and lipopolysaccharide-associated proteins, while E. aerogenes cells are affected through the disruption of bonds between KDO and the outer membrane.