Resuscitation of Salmonella enterica serovar typhimurium and enterohemorrhagic Escherichia coli from the viable but nonculturable state by heat-stable enterobacterial autoinducer

Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of S. enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the sigma(s) subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.     

A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila

Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.     

A Mixed Culture Recovery Method Indicates that Enteric Bacteria Do Not Enter the Viable but Nonculturable State

A new method, called the mixed culture recovery (MCR) method, has been developed to determine whether recovery of culturable bacterial cells from a population of largely nonculturable cells is due to resuscitation of the nonculturable cells from a viable but nonculturable state or simply to growth of residual culturable cells. The MCR method addresses this issue in that it involves the mixing of two easily distinguishable strains (e.g., lactose positive and negative) in such a way that large numbers of nonculturable cells of both strains are present together with a small number of culturable cells of only one strain, performing a nutrient addition resuscitation procedure, and then plating the cells to determine whether both cell types are recoverable. In repeated experiments with strains of Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter aerogenes, and Salmonella choleraesuis, only cells of the culturable strain were recovered after application of various resuscitation techniques. These results suggest that the nonculturable cells were dead and that the apparent resuscitation was merely due to the growth of the remaining culturable cells.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

Resuscitation of Salmonella enterica Serovar Typhimurium and Enterohemorrhagic Escherichia coli from the Viable but Nonculturable State by Heat-Stable Enterobacterial Autoinducer

Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of S. enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the σ^s subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.     

Entry of Vibrio harveyi and Vibrio fischeri into the viable but nonculturable state

AIMS: Physiological responses of marine luminous bacteria, Vibrio harveyi (ATCC 14216) and V. fischeri (UM1373) to nutrient-limited normal strength (35 ppt iso-osmolarity) and low (10 ppt hypo-osmolarity) salinity conditions were determined. METHODS AND RESULTS: Plate counts, direct viable counts, actively respiring cell counts, nucleoid-containing cell counts, and total counts were determined. Vibrio harveyi incubated at 22 degrees C in nutrient-limited artificial seawater (ASW) became nonculturable after approximately 62 and 45 d in microcosms of 35 ppt and 10 ppt ASW, respectively. In contrast, V. fischeri became nonculturable at approximately 55 and 31 d in similar microcosms. Recovery of both culturability and luminescence of cells in the viable but nonculturable state was achieved by addition of nutrient broth or nutrient broth supplemented with a carbon source, including luminescence-stimulating compounds. Temperature upshift from 22 degrees C to 30 degrees C or 37 degrees C did not result in recovery from nonculturability. CONCLUSIONS: The study confirms entry of V. harveyi and V. fischeri into the viable but nonculturable state under low-nutrient conditions and demonstrates nutrient-dependent resuscitation from this state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms loss of luminescence of V. harveyi and V. fischeri on entry into the viable but nonculturable state and suggests that enumeration of luminescent cells in water samples may be a rapid method to deduce the nutrient status of a water sample.     

Direct inoculation into media containing bile salts and antibiotics is unsuitable for the detection of acid/salt stressed Escherichia coli O157:H7

The efficiency of selective enrichment broths for the recovery of low numbers of acid/salt stressed Escherichia coli O157:H7 was determined. Stressed cultures were diluted to low levels and recovered in tryptone soya broth with added bile salts, to make modified tryptone soya broth, and buffered peptone water with various combinations of antibiotic supplementation including novobiocin, acriflavine and a mixture of vancomycin, cefsulodin and cefixime (VCC) at 37 °C and 42 °C. Significantly fewer stressed cells, in some cases as little as 0·3% of the starting population, were recovered by all the selective enrichment broths containing bile salts or VCC antibiotics compared to the non-selective controls. The use of such enrichments to recover low numbers of stressed E. coli O157:H7 may result in failure to detect the organism. Parallels with salmonella methodology are made and the need for a non-selective pre-enrichment stage in E. coli O157:H7 methods discussed.     

Nutrient shock and incubation atmosphere influence recovery of culturable Helicobacter pylori from water

Three different media-Columbia agar, Wilkins-Chalgren agar, and Helicobacter pylori special peptone agar-were prepared in a diluted version and compared to the standard medium formulation in order to study a possible nutrient shock effect observed when recovering H. pylori from water by counting the number of CFU. This same parameter was subsequently used to evaluate the influence of the incubation atmosphere by using a modular atmosphere-controlled system to provide different atmospheres and by employing an established gas generation kit as a control. Both a low nutrient content of the media and a rapidly achieved microaerophilic incubation atmosphere proved to increase the numbers of environment-stressed H. pylori organisms recovered. An atmosphere of 5% CO(2), 5% O(2), and 3% H(2) is recommended, although other atmospheres with a low oxygen concentration are also acceptable. Besides highlighting and assessing the importance of several factors in the culturability of H. pylori, this paper demonstrates the potential ability to develop an optimized technique for recovery of this pathogen from water.     

Resuscitation of Vibrio vulnificus from the Viable but Nonculturable State

Like many other gram-negative bacteria, the human pathogen Vibrio vulnificus is induced into a viable but nonculturable (VBNC) state by incubation at low temperatures. The ability of any bacterium to resuscitate from this dormant state would appear to be essential if the VBNC state is truly a survival strategy. The question as to whether the culturable cells which appear following removal of the inducing stress are a result of true resuscitation or of regrowth of a few residual culturable cells has long been debated. V. vulnificus was examined for its ability to resuscitate from this state following a temperature upshift. Several lines of investigation, including dilution studies, determination of the time necessary for appearance of a culturable population, and the effects of nutrient on recovery, all indicated that, at least for V. vulnificus, true resuscitation does occur. Our studies further suggest that nutrient is in some way inhibitory to the resuscitation of cells in the VBNC state and that studies which add nutrient in an attempt to detect resuscitation are able to detect only residual culturable cells which might be present and which were not inhibited by the added nutrient.     

Recovery of culturability of an HOCl-stressed population of Escherichia coli after incubation in phosphate buffer: resuscitation or regrowth?

An Escherichia coli population harvested in exponential phase at about 10(8) cells/ml was treated in phosphate buffer with HOCl at concentrations ranging from 0.4 to 1 mg/liter (7.7 to 19 microM). The HOCl stress resulted in the appearance of three cell subpopulations: a majority of dead (nonrespiring) cells, a few culturable cells (10(2) to 10(4)), and about 10(7) viable but nonculturable cells. In the absence of any added exogenous nutrient, a culturable population could be recovered after 1 day of incubation in phosphate buffer, and such a population would reach a cell density close to 10% of the initial density of the stressed population, whatever the initial number of survivors. When a small number of untreated cells were mixed with the stressed population, growth of the untreated cells was observed, demonstrating that damaged cells provided nutrients. Similarly, a filtrate and a disrupted-cell filtrate of the stressed population supported growth of untreated cells with the same efficiency. The number of CFU (untreated or stressed) at plateau phase depended on the initial density of the stressed cells. Taken together, these results suggest that recovery in phosphate buffer of an HOCl-stressed population is in large part due to growth of a few culturable cells at the expense of damaged cells. However, comparison of the growth rates of the stressed culturable population and of untreated bacteria growing in filtrate showed significantly faster growth of the stressed cells, a fact not fully compatible with the hypothesis that recovery is only the simple growth of survivors. We suggest, therefore, that in addition to growth of the few culturable stressed cells, there is repair and growth of some mildly injured viable but nonculturable cells.     

Use of autoradiography to assess viability of Helicobacter pylori in water.

Autoradiographic methods have been developed to detect metabolic activity of viable but nonculturable cells of Helicobacter pylori in water. Four strains of H. pylori were studied by using microcosms containing suspensions of 72-h cultures in water. The suspensions of aged, nonculturable cells of H. pylori were incubated with [3H]thymidine for 24 to 72 h, after which the cell suspensions were exposed to Kodak NTB2 emulsion for 3 to 28 days. Each sample was processed with three separate controls to rule out false-positive reactions. The organism remains viable and culturable under these conditions for up to 48 h and, in some cases, 20 to 30 days, depending on physical conditions of the environment. We found that temperature was a significant (P < or equal to 0.01) environmental factor associated with the viability of H. pylori cells in water. Autoradiographs of tritium-labeled cells of H. pylori revealed aggregations of silver grains associated with uptake by H. pylori of radiolabelled substrate. Findings based on the autoradiographic approach give strong evidence supporting the hypothesis that there is a waterborne route of infection for H. pylori. The possibility that H. pylori may persist in water in a metabolically active stage but not actively growing and dividing is intriguing and relevant to public health concerns.     

Performance of media for recovering stressed cells of Enterobacter sakazakii as determined using spiral plating and ecometric techniques

A study was done to determine the performance of differential, selective media for supporting resuscitation and colony development by stressed cells of Enterobacter sakazakii. Cells of four strains of E. sakazakii isolated from powdered infant formula were exposed to five stress conditions: heat (55 degrees C for 5 min), freezing (-20 degrees C for 24 h, thawed, frozen again at -20 degrees C for 2 h, thawed), acidic pH (3.54), alkaline pH (11.25), and desiccation in powdered infant formula (water activity, 0.25; 21 degrees C for 31 days). Control and stressed cells were spiral plated on tryptic soy agar supplemented with 0.1% pyruvate (TSAP, a nonselective control medium); Leuschner, Baird, Donald, and Cox (LBDC) agar (a differential, nonselective medium); Oh and Kang agar (OK); fecal coliform agar (FCA); Druggan-Forsythe-Iversen (DFI) medium; violet red bile glucose (VRBG) agar; and Enterobacteriaceae enrichment (EE) agar. With the exception of desiccation-stressed cells, suspensions of stressed cells were also plated on these media and on R&F Enterobacter sakazakii chromogenic plating (RF) medium using the ecometric technique. The order of performance of media for recovering control and heat-, freeze-, acid-, and alkaline-stressed cells by spiral plating was TSAP > LBDC > FCA > OK, VRBG > DFI > EE; the general order for recovering desiccated cells was TSAP, LBDC, FCA, OK > DFI, VRBG, EE. Using the ecometric technique, the general order of growth indices of stressed cells was TSAP, LBDC > FCA > RF, VRBG, OK > DFI, EE. The results indicate that differential, selective media vary greatly in their abilities to support resuscitation and colony formation by stressed cells of E. sakazakii. The orders of performance of media for recovering stressed cells were similar using spiral plating and ecometric techniques, but results from spiral plating should be considered more conclusive.     

Metabolic processes involved in repair of Escherichia coli cells damaged by exposure to acid mine water

Escherichia coli was stressed by exposure to filter-sterilized acid mine water. Synthetic processes required for repair of sublethally injured survivors were studied by the addition of specific metabolic inhibitors to a resuscitation broth. Inhibitors of protein, RNA, DNA, lipid, and peptidoglycan synthesis as well as uncouplers and inhibitors of electron transport and ATPase activity were used. Acid mine water injury was severe, causing damage to the outer and cytoplasmic membranes. Repair of sublethally injured cells required protein, RNA, and lipid synthesis as well as a proton motive force.     

Metabolic processes involved in repair of Escherichia coli cells damaged by exposure to acid mine water.

Escherichia coli was stressed by exposure to filter-sterilized acid mine water. Synthetic processes required for repair of sublethally injured survivors were studied by the addition of specific metabolic inhibitors to a resuscitation broth. Inhibitors of protein, RNA, DNA, lipid, and peptidoglycan synthesis as well as uncouplers and inhibitors of electron transport and ATPase activity were used. Acid mine water injury was severe, causing damage to the outer and cytoplasmic membranes. Repair of sublethally injured cells required protein, RNA, and lipid synthesis as well as a proton motive force.     

A recombinant bacteriophage-based assay for the discriminative detection of culturable and viable but nonculturable Escherichia coli O157:H7

A previously green fluorescent protein (GFP)-labeled PP01 virulent bacteriophage, specific to Escherichia coli O157:H7, was used to construct lysozyme-inactivated GFP-labeled PP01 phage (PP01e-/GFP). The new recombinant phage lacked lytic activity because of the inactivation of gene e, which produces the lysozyme responsible for cell lysis. Gene e was inactivated by inserting an amber stop codon. Prolonged incubation of E. coli O157:H7 cells with PP01e-/GFP did not lead to cell lysis, while the propagation of PP01e-/GFP in host cells increased the intensity of green fluorescence. Retention of cell morphology and increase in fluorescence enabled the direct visualization and enumeration of E. coli O157:H7 cells within an hour. The PP01e-/GFP system, when combined with nutrient uptake analysis, further allowed the discriminative detection of culturable, viable but nonculturable (VBNC), and dead cells in the stress-induced aquatic environment. Stress-induced cells, which retained culturability, allowed phage propagation and produced bright green florescence. Nonculturable cells (VBNC and dead) allowed only phage adsorption but no proliferation and remained low fluorescent. The low-fluorescent nonculturable cells were further differentiated into VBNC and dead cells on the basis of nutrient uptake analysis. The low-fluorescent cells, which grew in size by nutrient incorporation during prolonged incubation in nutrient medium, were defined as metabolically active and in the VBNC state. The elongated VBNC cells were then easily recognizable from dead cells. The proposed assay enabled the detection and quantification of VBNC cells. Additionally, it revealed the proportion of culturable to VBNC cells within the population, as opposed to conventional techniques, which demonstrate VBNC cells as a differential value of the total viable count and the culturable cell count.     

Maintenance of pathogenicity during entry into and resuscitation from viable but nonculturable state in Aeromonas hydrophila exposed to natural seawater at low temperature

AIMS: To investigate the fate of Aeromonas hydrophila pathogenicity when cells switch, in nutrient-poor filtered sterilized seawater, between the culturable and nonculturable state. METHODS AND RESULTS: Aeromonas hydrophila ATCC 7966, rendered non culturable within 50-55 days of exposure to marine stress conditions, was tested for its ability to maintain haemolysin and to adhere to McCoy cells. Results showed that pathogenicity was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by the Kogure cell elongation test. However, this loss is only temporary because, following temperature shift from 5 to 23 degrees C, multiple biological activities of recovered Aer. hydrophila cells, which include their ability to lyse human erythrocytes and to attach and destroy McCoy cells were regained. During the temperature-induced resuscitation, constant total cell counts were observed. Moreover, no significant improvement in recovery yield was obtained on brain-heart infusion (BHI) agar plates amended with catalase. We suggest that in addition to the growth of the few undetected culturable cells, there is repair and growth of some mildly injured viable but nonculturable cells. CONCLUSIONS: The possibility that nonculturable cells of normally culturable Aer. hydrophila in natural marine environment may constitute a source of infectious diseases posing a public health problem was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when Aer. hydrophila cells are released in natural seawater with careful attention to the conditions in which surrounding waters gradually become warmer in late summer/early autumn.     

The oxidative metabolism of thioglycollate-elicited mouse peritoneal macrophages: the relationship between oxygen, superoxide and hydrogen peroxide and the effect of monolayer formation

The oxygen and glucose metabolism of peritoneal macrophages harvested from untreated mice (resident cells) and mice given an i.p. injection of thioglycollate broth (thioglycollate cells) were examined. Thioglycollate cells consumed approximately 3 times as much O2 at rest and during phagocytosis as resident cells, but oxygen reduction products (superoxide and hydrogen peroxide) could be recovered in only minimal amounts despite triggering by phagocytosis or exposure to PMA. Indirect evidence for the formation of oxygen reduction products such as O2- by thioglycollate cells was obtained by observation of the major pathways for glucose oxidation and NBT dye reduction. When thioglycollate cells were allowed to adhere to a glass surface O2- and H2O2 were easily recovered in the extracellular medium with a 20-fold increase above cells in suspension exposed to PMA. This study suggests that thioglycollate-elicited macrophages have a vigorous oxidative metabolism but that recovery, and perhaps utilization, of O2 reduction products formed will depend on the conditions of incubation. These events may be significant both for the study of parameters of macrophage "activation" in vitro as well as the function of these cells in vivo.     

Interaction of amoxicillin with DNA in human lymphocytes and H. pylori-infected and non-infected gastric mucosa cells

Amoxicillin is a penicillin derivative belonging to a group of beta-lactam antibiotics used in Helicobacter pylori eradication. Clinical application of amoxicillin is underlined by its antibacterial activity, but little is known about its interaction with DNA of human cells. Using the alkaline comet assay we investigated the genotoxicity of amoxicillin in human peripheral blood lymphocytes as well as in H. pylori-infected and non-infected human gastric mucosa cells. To assess the role of reactive oxygen species in the genotoxicity of amoxicillin we employed a set of antioxidant and free radical scavengers, including Vitamins C and E, melatonin and the nitrone spin trap N-tert-butyl-alpha-phenyl-nitrone (PBN). Amoxicillin-induced DNA damage was completely repaired after 60 min. The vitamins, melatonin and the spin trap decreased the extent of the damage. The cells exposed to amoxicillin and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized bases displayed greater extent of DNA damage than those not treated with these enzymes. H. pylori non-infected gastric mucosa cells exposed to hydrogen peroxide repaired their DNA in a 60 min incubation, but the infected cells were not able to do so. The action of DNA repair enzymes, the vitamins, melatonin and PBN indicated that amoxicillin-induced oxidative DNA damage. The drug did not induce DNA strand breaks in isolated pUC19 plasmid DNA. Our results suggest that amoxicillin can induce DNA damage in human lymphocytes and gastric mucosa cells and this effect may follow from the production of reactive oxygen species. Cellular activation of the drug is needed to induce DNA damage. Free radical scavengers and antioxidants may be used to assist H. pylori eradication with amoxicillin to protect DNA of the host cells. Our results suggest also that H. pylori infection may alter gastric mucosa cells response to DNA-damaging agents and in this way contribute to initiation/promotion of cancer transformation of these cells induced by external or internal carcinogens.