APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

Oct. 1999, p. 4677–4681 Vol. 65, No. 10

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Viability and DNA Maintenance in Nonculturable SpiralCampylobacter jejuni Cells after Long-Term Exposure to

Low TemperaturesBEATRIZ LAZARO,1 JOSE CARCAMO,2 ANA AUDICANA,2 ILDEFONSO PERALES,2

AND AURORA FERNANDEZ-ASTORGA1*

Departamento de Inmunologıa, Microbiologıa y Parasitologıa, Facultad de Farmacia, Universidad del Paıs Vasco(UPV/EHU), 01080 Vitoria-Gasteiz,1 and Laboratorio Normativo de Salud Publica, Departamento de Sanidad,

Gobierno Vasco (GV/EJ), 48010 Bilbao,2 Spain

Received 7 June 1999/Accepted 10 July 1999

Survival of Campylobacter jejuni at 4 and 20°C was investigated by using cellular integrity, respiratoryactivity, two-dimensional (2D) protein profile, and intact DNA content as indicators of potential viability ofnonculturable cells. Intact DNA content after 116 days, along with cellular integrity and respiring cells, wasdetected for up to 7 months at 4°C by pulsed-field gel electrophoresis. Most changes in 2D protein profilesinvolved up- or down-regulation.

Campylobacter jejuni, a common food-borne enteropathogenin developed countries, is unable to multiply in foods, but itclearly survives in numbers sufficient to cause human disease.It is generally accepted that low temperatures enhance survivalof campylobacters (3), whereas high temperatures provokequick transformation from culturable spiral-shaped to noncul-turable coccoid forms. Given the importance of foods as vehi-cles of C. jejuni along with the extensive use of low tempera-tures for preservation, studies examining the survival of C.jejuni are required. Furthermore, the survival of bacterial cellsin cold environments could be for longer than that detected byculturability if viability is maintained in the absence of cultur-ability, i.e., if cells are in the so-called viable-but-nonculturable(VBNC) state. This state has been proposed previously for C.jejuni (19, 23), but resuscitation of putative VBNC cells inlaboratory animals (22, 23) has not always been reproducible(17). A number of methods based on maintenance of cellularstructures (10), metabolic activity (1, 14, 18, 23), and/or thepresence of nucleic acid (21, 25, 27) have been proposed toassess the viability of nonculturable cells, but at present, nonehas been agreed upon as being suitable overall. So, more thanone criterion must be taken into account for considering theviability of nonculturable cells (16). In the present study,change in total cell protein profile is also included to test theviability of C. jejuni nonculturable cells after exposure to ad-verse conditions, mainly nutrient depletion and low tempera-ture. The concurrence of spiral and/or coccoid forms in suchnonculturable cells is also discussed. Two C. jejuni strains wereused, a human isolate from the Hospital of Txagorritxu, Vito-ria-Gasteiz, Spain, designated C-1, and its derivative, C-1RR,obtained after passage twice through the mouse intestine.

Culture conditions and bacterial counts. For culture andlong-term incubation purposes, strains were grown on campy-lobacter agar base (Oxoid) supplemented with 5% lysed horseblood (Oxoid) for 24 h at 42°C, under a microaerobic atmo-sphere (7% CO2, 8% O2, and 85% N2). For survival experi-

ments, strains were grown in nutrient broth no. 2 (Oxoid) foran additional 24 h, harvested by centrifugation, suspended in500 ml of phosphate-buffered saline (PBS) (pH 7.3) at a finaldensity of 109 cells ml21, and then incubated without shakingin the dark at 4 and 20°C. At the time of inoculation and atregular intervals, culturability was assessed by standard platecounting and epifluorescence direct counts. Total bacterialcounts were microscopically performed by the standard acri-dine orange direct procedure (10). Metabolic activity was de-termined by tetrazolium salt reduction as an indication of anactive electron transport chain (18), and the number of respir-ing cells was determined by staining with 5-cyano-2,3-ditolyltetrazolium chloride according to the method of Cappelier etal. (4). Counts were the means of at least three determinations.Morphological changes and average dimensions of the C. jejunicells were monitored by computerized image analysis with PC-Image (Foster Findlay Assoc. Ltd.) with an Olympus epifluo-rescence microscope (BX40) equipped with a Sony DXC-950-P video camera.

Survival curves. Direct cell counts determined in parallelwith respiratory activity and culturability showed that the cel-lular integrity and respiratory activity were maintained muchlonger than culturability. In fact, survival continued for up to 7months based on signs of viability other than culturability.Changes in cell morphology from spiral to coccoid forms werealso detected (Fig. 1). At the beginning of the incubationperiod, C. jejuni cells from late log phase were mainly spiralcells with a stable average (95% confidence interval) length ofca. 1.4859 (1.4069 to 1.5649) mm. At the end of the incubationperiod, this average (95% confidence interval) length signifi-cantly decreased to ca. 1.2409 (1.1627 to 1.3191) mm at 4°C andca. 1.2925 (1.2253 to 1.3597) mm at 20°C. Electron microscopy(Fig. 2) revealed typical spiral rods with a single polar (orbipolar) flagellum and a relatively smooth surface and a fewspheroid cells with or without flagella.

Unlike findings for other curved bacilli such as Vibrio para-haemolyticus (13), C. jejuni and other campylobacter (11) cellsbecame spheroid more quickly when kept at room tempera-ture. In previous studies, the transition to nonculturable cellswas assumed to be associated with a morphological changefrom spiral to coccal shape (2, 17). However, our results do notsupport this assumption. The transition to coccoid form was

* Corresponding author. Mailing address: Departamento de Inmu-nologia, Microbiologia y Parasitologia, Facultad de Farmacia, Univer-sidad del Pais Vasco (UPV/EHU), Paseo de la Universidad, 7, 01006Vitoria-Gasteiz, Spain. Phone: 34 945 013909. Fax: 34 945 130756.E-mail: oipfeasa@vf.ehu.es.

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not always related to the decrease of culturability, as loss ofculturability occurred when only a third of the cells were coc-coid forms. Furthermore, within the first 30 days of starvationat 4°C, the percentage of respiring cells was higher than thoseof either spiral or culturable cells, features which were alsonoted with the C-1 strain after 5 days of incubation at 20°C.Such results could be explained if the spiral forms of C. jejunicells are in either the culturable or the nonculturable physio-logical state and if cells other than culturable spiral forms arealso active respiring cells. Some of these results are in agree-ment with those reported for the closely related species Heli-cobacter pylori (15). There is, however, a major difference;while Kusters et al. (15) propose the loss of culturability as aloss of viability, we observe the viability of these nonculturablecells on the basis of their potential for respiration as well ascellular integrity (and other characteristics, discussed below) inspite of their spiral or coccoid morphology. Therefore, ourdata indicates that two forms of nonculturable C. jejuni cellsmight exist: viable and nonviable, which might not correspondwith spiral and coccoid forms, respectively.

Strain influence was found mainly on the basis of the highestpercentages of spiral cells detected for C-1RR long after non-culturability was reached during incubation at 20°C. It seemslikely that the origin of the strain may influence the rate ofmorphological change. There is evidence to suggest that theadaptation to the intestinal tract enhances the ability of strainsto colonize (5) or to be virulent (20). Our results suggest thatadaptation to the mouse intestine enhances the maintenanceof the spiral shape during starvation at room temperature.

Bleb-like membrane vesicles were also observed around thecells incubated at 4°C. Formation of these visible excrescences,

possibly formed by pieces of cell envelope, is known to occur inother curved bacilli such as Vibrio cholerae (12), V. parahae-molyticus (13), and H. pylori (15) during starvation. We agreewith the explanation of Jiang and Chai (13) for these features.Instead of degenerative forms as proposed by Kusters et al.(15), these vesicles could be due to a process of cell volumeadjustment by bleb formation representing a survival strategyfor minimizing cell maintenance requirements and enhancingsubstrate uptake due to a high surface-volume ratio. It wasproposed by McDougald et al. (16) that changes in cell wallsand cell membranes allow for long-term stability and survivalof the bacterial cells. Our data supports these assumptionsbecause we found blebs and statistically significant (P #0.0001) cell size reduction as well as maintenance of spiralmorphology during incubation of C. jejuni cells at low temper-ature.

Two-dimensional gel electrophoresis. To further character-ize the nonculturable cells, protein profiles from crude cellextracts were analyzed by two-dimensional gel electrophoresis(Fig. 3C and D) and compared with profiles at day 0 (Fig. 3Aand B) for both strains, C-1 and C-1RR. In controls, five majorcharacteristic bands were readily identified and labeled as pro-teins 1, 2, 3, 4, and 5. Several other protein bands (arrows inFig. 3) were usually observed in both strains, but their visual-ization was silver stain dependent, and that made comparisonsdifficult. It is noteworthy that all of the C. jejuni strains assayed(not all shown here) had remarkable similarity in two-dimen-sional profiles. The major outer membrane protein was clearlyidentified as the protein of about 40 kDa (protein 4). It waspredominant in all cases, but its relative amount decreasedwith increased time of incubation at either temperature. The

FIG. 1. Survival curves of C. jejuni C-1 during incubation in PBS at 4°C (A) and 20°C (B) and of its derivative C-1RR at 4°C (C) and 20°C (D). Initial numbers ofrespiring cells in experiments at 4°C were 9.77 3 108 and 1.23 3 109 ml21 and numbers of culturable cells were 3.14 3 108 and 2.3 3 109 CFU ml21 for strains C-1and C-1RR, respectively. At 20°C, initial numbers of respiring cells were 1.22 3 108 and 1.02 3 109 ml21 and the numbers of culturable cells were 1.85 3 108 and 1.05 3109 CFU ml21 for strains C-1 and C-1RR, respectively. Points are mean values from triplicate determinations.

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62-kDa protein (protein 2) was identified as flagellin, and the14-kDa protein (protein 5) could be the same as that reportedby Wu et al. (26).

Differences in the protein profiles of culturable and noncul-turable cells of C. jejuni were also detected at both tempera-tures. Most of them could be explained by up- or down-regu-lation mechanisms except for protein 7 (60 kDa), which wasalways detected after long-term incubation (196 days) at 20°C,without apparent reduction of expression of the 40-kDa and62-kDa major proteins. Similar events have been describedpreviously for Vibrio anguillarum (7) in response to growth atadverse temperatures. Culture conditions were quite different,and those authors reported a great decrease of the 40-kDamajor outer membrane protein with an increase of the 60-kDaprotein. Another possible explanation is that some of the mainprotein components were degenerating and modifying theirisoelectric point. If so, a great reduction of expression of atleast one such protein should have been detected, but this wasnot the case. Therefore, in the absence of more detailed in-

formation, this 60-kDa protein must have a different origin andrequires future investigation. It must be remembered that denovo protein synthesis below the minimal growth temperaturehas been observed previously for C. jejuni (9).

DNA maintenance. Restriction endonuclease digestion andpulsed-field gel electrophoresis (PFGE) were carried out todetermine whether the DNA content was maintained intactover the long-term incubation at both temperatures. The ex-periments were carried out with cells of strain C-1, incubatedin PBS at 4 and 20°C. Survival sampling was done at thebeginning of incubation (as control) and long after cells hadbecome nonculturable. PFGE patterns (Fig. 4) with SalI andSmaI were determined because these restriction endonucle-ases were used by Chang and Taylor (6). The number of frag-ments we obtained was almost identical with those reported byChang and Taylor (6).

Digested DNA from controls displayed the same PFGEprofile in each run, in spite of the initial number of cells,indicating that the PFGE protocol generated reproducible re-

FIG. 2. Transmission electron micrographs showing the different morphologies of C. jejuni cells at several times during incubation at 4°C. Bacterial cells were viewedon 300-mesh grids by transmission electron microscopy (Philips CM10 microscope) following negative staining with 2% (wt/vol) potassium phosphotungstate. (A)Typical spiral cell; (B and C) coccoid and concomitant spiral and coccoid forms showing the presence of the flagella; (D and E) cells showing bleb formation (arrows).

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sults (Fig. 4, lanes a to c). Because of the difference in detec-tion levels of the bands, 5 3 109 spiral cells ml21 were requiredfor DNA extraction, and even-higher numbers were requiredwhen cells became coccoid. PFGE profiles from incubations at4°C were easily compared, but those from incubations at 20°Cwere difficult to resolve despite the higher number of cellslysed. Decreases in DNA detectability may be attributable notonly to the progressive loss and/or degradation of DNA butalso to the cell envelope alteration and/or lower efficiency ofcell lysis. Cells which enter the VBNC state undergo changeswhich allow them to survive in the environment for extendedperiods (16). These changes, observed for several organismsincluding C. jejuni (19), involve alterations of the compositionof the cell wall and cell membrane (and thereby of function)but do not affect cellular or DNA integrity.

Nonculturable cells after 116 days in PBS at 4°C (Fig. 4,lanes e and i) maintained intact chromosomal DNA andyielded easily recognizable bands in agarose gels producing thesame profiles as fresh culturable cells from day 0 (Fig. 4, lanesa to c and g). During incubation at 20°C, bands in agarose gelsbecame nearly undetectable. Even so, in profiles of cells from20 and 61 days at 20°C (Fig. 4, lanes d, h, k, and l) PFGEbanding patterns were the same as those from control cells aswell as those from cells kept at 4°C. Although the PFGEpatterns are relatively stable, bacteria with small genomes,such as C. jejuni, may undergo genetic variation to increase

FIG. 3. Two-dimensional, silver-stained protein profiles of C. jejuni cells. Profiles are shown for culturable cells of strain C-1 (A) and strain C-1RR (B) from latelog phase at day 0 (control) and for nonculturable cells of strain C-1 after 196 days of incubation in PBS at 4°C (C) and 20°C (D). Isoelectric focusing was performedwith a gel containing 4.1% (vol/vol) acrylamide and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 12.5% (wt/vol) polyacrylamide gel (MiniProtean II two-dimensional system; Bio-Rad). A low-range sodium dodecyl sulfate-polyacrylamide gel electrophoresis standard (14.4 to 97.4 kDa; Bio-Rad) was runin all the gels. Individual proteins that were usually observed in both strains are labeled with arrows. Numbers 1, 2, 3, 4, 5, and 6 show major characteristic protein bands.Number 7 denotes an up-regulated protein after 196 days at 20°C.

FIG. 4. PFGE of DNA extracted from C. jejuni and digested with SalI (lanesa to e and k) and SmaI (lanes g to i and l). The restriction fragments wereseparated in a 1.2% agarose gel by electrophoresis for 24 h at 170 V and 14°C,with ramped pulse times from 10 to 35 s (contour-clamped homogeneous electricfield DR II PFGE apparatus; Bio-Rad). After electrophoresis, the gels werestained with ethidium bromide. Lanes: a to c, strain C-1 at day 0 of starvation at1 3 109, 5 3 109, and 1 3 1010 cells ml21, respectively; d, strain C-1 at day 20 ofstarvation at 20°C; e, strain C-1 at day 116 of starvation at 4°C; f, l DNA ladderranging from 48.5 to 485 kb; g, strain C-1 at day 0 of starvation at 5 3 109 cellsml21; h, strain C-1 at day 20 of starvation at 20°C, i, strain C-1 at day 116 ofstarvation at 4°C; j, strain C-1 at day 116 of starvation at 4°C and not digested;k and l, strain C-1 at day 61 of starvation at 20°C.

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their potential to adapt to new environments (8, 24), and so thevariation in PFGE genotype may be attributable to genomicvariation. It has been reported previously for Vibrio vulnificus(25) and Legionella pneumophila (27) that prolonged exposureof cells to cold leads to a gradual degradation of DNA andRNA in an increasing fraction of the population, while a smallsubpopulation that maintains intact nucleic acids may retainviability. Because no changes in the recognition sequence weredetected, PFGE genotypes from our strain were stable after 4and 2 months of incubation at 4 and 20°C, respectively (morethan 1 month after becoming nonculturable). These findingsmay be compatible with viability in such cells. Therefore, weagree with authors who consider these nonculturable cellsmaintaining intact DNA to be VBNC cells. This conversion toVBNC forms and the transition to coccoid forms are twodifferent but related phenomena. Coccoid forms could corre-spond to the second phase of conversion proposed by Weichartet al. (25) in the formation of VBNC cells, and thus the realVBNC forms of C. jejuni could be found among the spiralnonculturable cells that maintain cellular integrity along withintact DNA. However, the inability to isolate exclusively coc-coid forms makes this difficult to prove.

This work was supported by Education, University and InvestigationDepartment grant PI95/40 from the Basque Government.

We thank Lourdes Michaus from the Txagorritxu Hospital for pro-viding some of the strains. We thank Susan Barrow for technicalassistance with the English language.

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