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Applied and Environmental Microbiology, August 2003, p. 4975-4978, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4975-4978.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Description of a "Phoenix" Phenomenon in the Growth of Campylobacter jejuni at Temperatures Close to the Minimum for Growth

A. F. Kelly, A. Martínez-Rodriguez, R. A. Bovill,{dagger} and B. M. Mackey*

School of Food Biosciences, University of Reading, Whiteknights, Reading RG6 6BZ, United Kingdom

Received 29 October 2002/ Accepted 12 May 2003


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ABSTRACT
 
When Campylobacter jejuni cultures that had been grown in broth at 39°C were subcultured into fresh medium at 30°C, there was a transient period of growth followed by a decline in viable-cell numbers before growth resumed once more. We propose that this complex behavior is the net effect of the growth of inoculum cells followed by a loss of viability due to oxidative stress and the subsequent emergence of a spontaneously arising mutant population that takes over the culture.


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INTRODUCTION
 
Campylobacter jejuni is now recognized as one of the more important bacterial causes of diarrheal illness in humans (15). In the United States, Campylobacter is estimated to cause more than 2 million cases of gastrointestinal illness annually (10), while in the United Kingdom cases of Campylobacter enteritis now exceed those due to Salmonella (http://www.phls.co.uk). Although the disease is normally self-limiting, complications such as reactive arthritis and Guillain-Barré syndrome may occur (16). Poultry meat, raw milk, pets, and untreated water are believed to be vehicles of infection, but our understanding of the routes of infection is far from complete (16). One particular puzzle is that the organism seems poorly adapted to survive outside its animal or avian host. It grows best under microaerobic conditions, is sensitive to environmental stresses such as drying, heating, or acidification, and has an unusually narrow temperature range for growth (15).

The upper and lower limits for growth of C. jejuni are reported to be around 30 and 47°C, respectively (4). Although multiplication ceases below 30°C, considerable metabolic activity can be detected in cells at temperatures as low as 15°C (5). Alterations in membrane fatty acid composition have been detected in cells incubated at low temperatures (6, 7), but in general, very little is known of the mechanisms by which C. jejuni adapts to changes in temperature or other environmental conditions. During investigations of the growth of C. jejuni at temperatures close to the minimum, we observed an unusual pattern of growth that is described here.


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Organisms and growth conditions.
 
C. jejuni NCTC 11351 (type strain) and C. jejuni strain 81116 (NCTC 11828) were stored at -70°C in Microbank vials (Pro-Lab Diagnostics, Neston, United Kingdom). Inoculum cultures were prepared as follows. A frozen bead was inoculated into 50 ml of brucella broth (Difco, East Moseley, United Kingdom) containing one vial of FBP Campylobacter growth supplement per 500 ml (SR84; Oxoid, Basingstoke, United Kingdom) (BBFBP) in a 100-ml flask. FBP Campylobacter growth supplement is a nonselective supplement containing sodium pyruvate, sodium metabisulfite, and ferrous sulfate to protect against oxidative stress. This culture was then incubated at 39°C on a shaking platform at 150 rpm for 24 h under microaerobic conditions (5% [vol/vol] oxygen, 10% [vol/vol] carbon dioxide, and 85% [vol/vol] nitrogen) maintained with a variable-atmosphere incubator (Don Whitely, Otley, United Kingdom). The resultant stationary-phase cells were then diluted 1:100 into 50 ml of fresh BBFBP and grown for 6 or 24 h to produce exponential- or stationary-phase cells, respectively.

To investigate growth at 30°C, cultures were grown in 100-ml bottles containing 60 ml of BBFBP. The bottles were sealed with a lid lined with a double gasket layer of autoclavable neoprene rubber. The lid contained three entry ports. The medium was sparged with a microaerobic gas mix (as described above) filtered through a sterile 0.22-µm-pore-size filter (Millipore, Watford, United Kingdom). Two sterile hypodermic needles inserted through the gasket layer allowed gas to be introduced into and vented from the vessel. The bottle assemblies were equilibrated at 30°C in a reciprocating water bath (90 rpm) for at least 1 h before inoculation. The bottles of broth medium were inoculated with ca. 105 CFU of exponential-phase cells/ml or 106 CFU of stationary-phase cells/ml. Samples were removed at intervals for viable-cell counting with a third hypodermic needle.


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Viable-cell counts.
 
Serial decimal dilutions were prepared in maximum-recovery diluent (Oxoid), and 20-µl volumes were spread onto fresh BMHA plates, consisting of Mueller-Hinton agar containing 5% defibrinated sheep's blood (TCS, Basingstoke, United Kingdom) and one vial of FBP Campylobacter supplement per 500 ml. The number of CFU was assessed after the plates had been incubated at 39°C in the variable-atmosphere incubator for a minimum of 48 h. The experiments were repeated two or more times, and representative results are shown.


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Production of spent broth.
 
Cultures of C. jejuni NCTC 11351 were grown to stationary phase at 37°C in 200 ml of BBFBP contained in a 500-ml flask in an anaerobe jar under microaerophilic conditions and then stored in the dark for 4 weeks. The cells were removed by centrifugation, and the supernatant was sterilized by filtering it through a 0.1-µm-pore-size filter (Nalgene, Loughborough, United Kingdom)


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Growth behavior of C. jejuni at temperatures close to the minimum for growth.
 
When stationary-phase cells of C. jejuni NCTC 11351 that had been grown at 39°C were inoculated into BBFBP at 30°C, there was an extended lag phase during which a complex but characteristic pattern of increasing and decreasing counts was observed. Typically, there was an immediate but short-lived period of growth followed by a much larger decrease in viable-cell numbers (Fig. 1). After continued incubation, the viable-cell count reached a minimum and growth then resumed once more (Fig. 1). This behavior was also seen with an inoculum of exponential-phase cells and with a stationary-phase inoculum incubated at the lower temperature of 29°C (Fig. 1). A similar pattern of growth at 30°C was also seen after inoculation with stationary-phase cells of a different strain, NCTC 11828 (data not shown).



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  		FIG. 1. Growth patterns of C. jejuni NCTC 11351 grown at 39°C in BBFBP and then inoculated into fresh medium at temperatures near the minimum for growth. Symbols: {blacktriangleup}, exponential-phase inoculum, 30°C; {square}, stationary-phase inoculum, 30°C; {circ}, stationary-phase inoculum, 29°C.

There was some variation in the pattern described above. The initial increase was seen in 10 out of 15 experiments in which C. jejuni was grown in BBFBP at 39°C and subcultured into fresh medium at 30 or 29°C. The extent of the increase varied between 0.11 and 0.62 log units (mean, 0.32 log unit). In five experiments, this initial increase was not observed and the count remained the same or declined during the first 24 h (the decreases ranged from 0.01 to 0.43 log unit; mean, 0.23 log unit). The net decrease in count from the inoculum level ranged from 0.75 to 3.21 log units with a mean of 1.99 log units. The time between inoculation and resumption of growth after the decline phase ranged from 72 to 270 h (mean, 128 h).


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Effect of spent medium and reduced oxygen tension on growth at 30°C.
 
Previous work (1) has shown what appeared to be a resuscitation of nonculturable cells of C. jejuni present in aged stationary-phase cultures. Further investigation of this phenomenon by use of the most-probable-number technique showed that most of the increase in viable-cell numbers could be explained by the rapid multiplication of residual culturable cells that were able to grow in the spent medium (1). To test whether spent medium might contain growth-stimulatory factors for stressed cells, we compared the growth patterns in fresh and spent broth at 30°C. In contrast to the behavior seen after inoculation into fresh broth, growth in spent broth at 30°C always occurred within 24 h and without loss of viability (Fig. 2). If diluted 1:3 with fresh medium, the spent medium was not effective in shortening the lag time. The extended lag phase at 30°C was also eliminated by sparging fresh growth medium with a microaerophilic gas mixture containing a lower concentration of oxygen (0.5% rather than 5%) (Fig. 2).



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  		FIG. 2. Effect of spent broth and reduced oxygen tension on growth of C. jejuni NCTC 11351 at 30°C. Growth in fresh BBFBP sparged with the microaerophilic gas mixture containing either 5% oxygen (•) or 0.5% oxygen ({blacksquare}) is shown as well as growth in spent BBFBP sparged with a 5% gas mixture ({square}).


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Effect of subculturing into fresh medium at 30°C.
 
To determine whether cells that eventually grew at 30°C had somehow adapted to that temperature, a fresh batch of BBFBP at 30°C was inoculated with cells taken from the stationary phase of a culture previously grown at 30°C (Fig. 3). In this case, growth occurred with little or no lag (Fig. 3). The absence of lag at 30°C might have been due to a physiological adaptation in cells grown at low temperature or to selection of a mutant strain that was better able to cope with low-temperature conditions. To test which of these possibilities was more likely, an inoculum of cells taken from the stationary phase of a culture grown at 30°C was subcultured at 39°C and grown for 18 h before inoculation into medium at 30°C. Even after growth at 39°C, such cells were able to commence growth at 30°C with no obvious lag or loss of viability (Fig. 3). This supported the view that cells in stationary-phase cultures that were able to commence growth rapidly at 30°C without loss of viability might have arisen by mutation. Next, single colonies were picked at random and purified from three separate stationary-phase cultures grown at 30°C. Each of these independent isolates was able to grow at 30°C without the extended lag normally seen at this temperature (Fig. 4). One of the isolates, designated SL1, was retained for further examination.



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  		FIG. 3. Effect of subculturing on growth of C. jejuni NCTC 11351 at 30°C. Symbols: •, original culture grown at 30°C; {blacksquare}, stationary-phase cells from original culture inoculated directly into fresh medium at 30°C; {square}, inoculum taken from original culture and then grown at 39°C before inoculation into medium at 30°C.



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  		FIG. 4. Growth at 30°C of single colony isolates obtained from three independent cultures of C. jejuni NCTC 11351 grown at 30°C to stationary phase. Strains SL1 ({circ}), SL2 ({blacksquare}), SL3 (•), and the parent strain ({blacklozenge}) were grown in BBFBP at 39°C and then subcultured into fresh medium at 30°C.


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Properties of variant strain SL1.
 
SL1 had the same biochemical profile as that of NCTC 11351 in the API Campy test kit (BioMérieux, Basingstoke, United Kingdom). SL1 was nonmotile when observed microscopically and when tested by growth from stab inocula in BBFBP plus 0.4% agar (3), but examination of negatively stained preparations by electron microscopy revealed the presence of polar flagella (data not shown). When grown on BMHA at 39°C, colonies of SL1 measured by image analysis were significantly smaller (p < 0.001) than those of NCTC 11351 (mean colony sizes after 48 h of incubation, 0.98 ± 0.07 mm [mean ± standard deviation] and 1.4 ± 0.09 mm, respectively). The basis of this colony phenotype is unknown but may be associated with nonmotility since similar changes in colony appearance have previously been noted for nonmotile Campylobacter coli (12). When grown on BMHA at 30°C, colonies of SL1 were visible after about 3 days rather than the usual 7 days necessary with the parent. There were no obvious differences between SL1 and NCTC 11351 in microaerobic growth rates at 37°C or in sensitivity to atmospheric oxygen at 39 or 30°C when measured as described by Kelly et al. (8) (data not shown).


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Interpretation of the phenomenon.
 
The growth pattern described here is very reminiscent of the Phoenix growth phenomenon first described for Clostridium perfringens grown at 50°C (2). This phenomenon was characterized by a decrease in viable-cell numbers immediately after inoculation, followed by an increase to the level of the initial count and a subsequent continued increase beyond the inoculum-level count. In later work (13), the initial decrease and increase in count were shown to be caused by an injury-and-recovery process that could be eliminated by using strictly anaerobic conditions during dilution and plating.

In C. jejuni, the phenomenon also seems to be related to oxidative stress because reducing the oxygen tension in the sparging gas eliminated the extended lag phase and loss of viability at 30°C. However, the effect seems to occur during growth rather than during dilution because we were unable to eliminate the effect by performing dilutions microaerobically rather than aerobically (data not shown). Also, unlike the case with Clostridium perfringens, the loss of viability in C. jejuni was often preceded by a short period of growth. It is possible that when the cells attempt to grow at 30°C, they enter an unbalanced physiological state in which the rate of generation of toxic oxygen species overwhelms the capacity of the cell to deal with these damaging agents. Spent medium was also protective, but the basis of this is unknown. This protective effect seems unlikely to be due to a specific cell signaling effect but may be associated with nonspecific protective or conditioning effects of components of spent medium. Reduced glutathione and other thiol compounds accumulate in stationary-phase cultures of Escherichia coli and Salmonella (11), and such compounds would be candidates to explain the protective effect observed here. However, whether similar compounds are found in cultures of Campylobacter spp. is unknown.

Because the short-lag phenotype of cells grown at 30°C was retained during subculture at 39°C, the most likely interpretation is that the cultures were taken over by mutants better able to tolerate low temperatures. This is evidently not a rare event, because such putative mutants were isolated independently from three separate cultures. The sequence of events giving rise to the pattern of growth shown in Fig. 1 might thus be an initial period of growth at 30°C that could not be sustained, followed by the death of a large proportion of the population and an eventual overgrowth by a spontaneously arising mutant that grew with little or no lag. The point during growth at which the putative mutants arose is not known, but lines extrapolated back from the (second) exponential growth phase of four growth curves of the type shown in Fig. 1 intersected the cell concentration axis at a point between 1 and 100 cells per 60-ml culture. There are of course many errors and assumptions in making such extrapolations, but the observation would be consistent with one or more of the relevant mutants being present in the inoculum. Further work will be required to characterize the mutants and determine their origin.

The genome of C. jejuni NCTC 11168 contains many hypervariable sequences that may be a source of genetic variability (13). Recently evidence has been presented that stationary-phase cultures of campylobacters are dynamic populations in which mutants frequently arise and supplant the original population (8). It has been proposed that this might be a general strategy to promote survival. The work here is consistent with this idea and suggests that mutation may also play a role in the growth and survival of campylobacters exposed to unfavorably low temperatures.

A further practical implication is suggested by the work of Miller et al. (9), who showed that some single colonies obtained by streaking out mixed cultures of C. jejuni contain more than one strain. If physiological variants can arise at a relatively high frequency in stressed populations as proposed here, it is possible that some colonies would contain more than one genotype and one phenotype, which could be a complicating factor in survival studies.


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ACKNOWLEDGMENTS
 
We are grateful to the Food Standards Agency/Ministry of Agriculture Fisheries and Food, London, United Kingdom, for financial support of this work. A.M.-R. was the recipient of a Marie Curie Individual Fellowship of the European Union.

We thank B. E. B. Moseley for many helpful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Food Biosciences, University of Reading, P.O. Box 226, Whiteknights, Reading RG6 6BZ, United Kingdom. Phone: 44 118 935 7229. Fax: 44 118 935 7222. E-mail: b.m.mackey{at}reading.ac.uk. Back

{dagger} Present address: Oxoid Ltd., Basingstoke, United Kingdom. Back


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Applied and Environmental Microbiology, August 2003, p. 4975-4978, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4975-4978.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




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