ORIGINAL PAPER

CadF expression in Campylobacter jejuni strains incubatedunder low-temperature water microcosm conditions whichinduce the viable but non-culturable (VBNC) state

Vania Patrone • Raffaella Campana • Luciana Vallorani •

Sabrina Dominici • Sara Federici • Lucia Casadei •

Anna Maria Gioacchini • Vilberto Stocchi • Wally Baffone

Received: 24 October 2012 / Accepted: 5 January 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Campylobacter jejuni is a major gastroin-

testinal pathogen that colonizes host mucosa via

interactions with extracellular matrix proteins such

as fibronectin. The aim of this work was to study

in vitro the adhesive properties of C. jejuni ATCC

33291 and C. jejuni 241 strains, in both culturable and

viable but non-culturable (VBNC) forms. To this end,

the expression of the outer-membrane protein CadF,

which mediates C. jejuni binding to fibronectin, was

evaluated. VBNC bacteria were obtained after

46–48 days of incubation in freshwater at 4 �C. In

both cellular forms, the expression of the cadF gene,

assessed at different time points by RT-PCR, was at

high levels until the third week of VBNC induction,

while the intensity of the signal declined during the

last stage of incubation. CadF protein expression by

the two C. jejuni strains was analysed using 2-dimen-

sional electrophoresis and mass spectrometry; the

results indicated that the protein, although at low

levels, is also present in the VBNC state. Adhesion

assays with culturable and VBNC cells, evaluated on

Caco-2 monolayers, showed that non-culturable bac-

teria retain their ability to adhere to intestinal cells,

though at a reduced rate. Our results demonstrate that

the C. jejuni VBNC population maintains an ability to

adhere and this may thus have an important role in the

pathogenicity of this microorganism.

Keywords Campylobacter jejuni � VBNC � cadF

gene expression � RT-PCR � CadF 2-DE analysis �CadF MS analysis

Introduction

Campylobacter jejuni, a Gram-negative, microaero-

philic, motile and spiral-shaped bacterium, is the most

common cause of food- and water-borne illness

worldwide (Butzler 2004). Infection by C. jejuni is

often associated with consumption of contaminated

poultry meat (Young et al. 2007) and produces

symptoms ranging from a mild, non-inflammatory,

watery diarrhoea to severe abdominal cramps, bloody

V. Patrone � R. Campana � S. Federici � W. Baffone (&)

Division of Toxicology, Hygienic and Environmental

Sciences Department of Biomolecular Sciences,

University of Urbino ‘‘Carlo Bo’’, Via S. Chiara 27,

61029 Urbino, Italy

e-mail: wally.baffone@uniurb.it

L. Vallorani � L. Casadei � A. M. Gioacchini � V. Stocchi

Division of Sport Science and Health, Department

of Biomolecular Sciences, University of Urbino

‘‘Carlo Bo’’, Via I Maggetti 26, 61029 Urbino, Italy

S. Dominici

Division of Biochemistry and Molecular Biology,

Department of Biomolecular Sciences, University of

Urbino ‘‘Carlo Bo’’, Via Saffi 2, 6129 Urbino, Italy

123

Antonie van Leeuwenhoek

DOI 10.1007/s10482-013-9877-5

diarrhoea, bacteraemia and death in the immunocom-

promised (Snelling et al. 2005). The pathogenic

processes that lead to the development of disease are

poorly understood (Dorrell and Wren 2007). Viru-

lence factors that contribute to the pathogenesis of

C. jejuni are associated with adaptation to the gut

environment, adherence to intestinal epithelial cells,

followed by internalisation, invasion, iron acquisition,

toxin production and alteration of host cell signalling

pathways, leading to host cell death (Ketley 1997).

Among the surface-exposed structures implicated in

bacterial adherence, C. jejuni possesses a 37 kDa

adhesin, termed CadF (Konkel et al. 1997, 1999, 2005)

that binds the extracellular matrix component fibro-

nectin (Konkel et al. 1997) and promotes bacteria–host

cell interactions (Konkel et al. 1997, 1999; Monteville

et al. 2003). The cadF gene coding this protein is

conserved among diverse groups of Campylobacter

spp. (Konkel et al. 1997, 1999).

When C. jejuni encounters environmental stressors,

such as nutrient starvation, osmotic shock and fluctu-

ations in temperature and pH, it can enter a viable but

non-culturable (VBNC) state (Rollins and Colwell

1986; Korhonen and Martikainen 1991) that repre-

sents a dormant form improving the survival of non-

sporulating bacteria in adverse environments (Oliver

1993; Colwell and Huq 1994; Barer and Harwood

1999). This state is physiologically important as it

allows survival until environmental conditions

become favourable for growth and cell division.

Although it is known that environmental stressors

can promote virulence in some pathogens, this pheno-

menon has not been well documented in C. jejuni

(Ma et al. 2009). The possibility that VBNC bacteria

can maintain their ability to adhere to living substrates

can be considered significant with regard to the first

essential step in the initiation of the infectious process

(Pruzzo et al. 2002).

In this study we have performed a molecular

analysis of the time-course of cadF gene expression

and applied two dimensional electrophoresis (2-DE)

and mass spectrometry (MS) analyses for CadF

protein levels in two C. jejuni strains during entry

into the VBNC state under conditions resembling

those found in natural freshwater environments. A

phenotypic analysis was also carried to evaluate the

adhesion ability of the resulting non-culturable forms

in comparison to culturable cells.

Materials and methods

Bacterial strains and growth conditions

The reference strain C. jejuni ATCC 33291 and a

human clinical isolate C. jejuni 241, both harbouring

the cadF gene as previously assessed in our laboratory

by specific primer PCR, were used for the experiments.

Bacterial strains were grown on Columbia Agar Base

(Oxoid, Milan, Italy) plates containing 5 % of Laked

Horse Blood (Oxoid) and the appropriate amount of

Preston Campylobacter Selective Supplement (Oxoid)

for 48 h at 42 �C under microaerophilic conditions (O2

5 %, CO2 10 %, N2 85 %). Bacteria were stored in

culture broth with 15 % of glycerol at -80 �C.

Production of VBNC cells and bacterial counts

The production of cells in the VBNC state and bac-

terial counts were performed as described previously

(Baffone et al. 2006), but inoculating the mid-loga-

rithmic phase suspension in freshwater. Culturable

counts (cfu/ml) were performed to assess the entry into

the VBNC state.

Double staining (CTC-DAPI) for viable (i.e. respir-

ing) and total cells counting was performed as

described by Rodriguez et al. (1992) every 3 days

until entry the VBNC state (\0.1 cfu/ml). To stimulate

cell respiration, 100 ll of a 0.05 g/l solution of pyruvic

acid (Sigma, Milan, Italy) were added to 1 ml of

microcosm sample (Cappelier et al. 1997); after that,

CTC (Polysciences, Trimital, Milan, Italy) was added

to a final concentration of 5 mM from a stock solution

in water. After incubation in the dark at 37 �C for 4 h,

cells were harvested by filtration through 0.22 lm

pore-size black polycarbonate membrane filters (Mil-

lipore, Milan, Italy) and then counterstained for 5 min

with 5 lg/ml DAPI (4,6-diamidino-2-phenylindoldi-

hydro chloride, Sigma) solution. Filters were air-dried,

mounted on glass microscope slides for fluorescence

and observed by Axiolab light microscope (Carl Zeiss

SpA., Milan, Italy). All the bacteria of the sample are

stained blue by DAPI and only active bacteria able to

reduce CTC show intracellular red fluorescent precip-

itate formazan crystals. Two filters for each sample

were counted and results are expressed as the number

of corresponding bacteria per ml of the initial inoculum

(Baffone et al. 2006).

Antonie van Leeuwenhoek

123

RT-PCR detection of the cadF gene in viable,

culturable and VBNC populations of C. jejuni

Total RNA extraction

Fifty milliliter water samples from each C. jejuni

microcosm were collected at 0 (T0), 7 (T7), 21 (T21),

35 (T35) and 46 (T46) days of incubation and filtered

through 0.22-lm membrane filters (Millipore, Vimod-

rone, Milan, Italy). One ml of a 1:2 solution of PBS-

RNAprotect Bacterial Reagent (Quiagen, Milan, Italy)

was added to the filters and vortexed for 60 s. The

bacterial suspension was incubated for 5 min at room

temperature and centrifuged at 15,000 rpm for 15 min

at 4 �C. The pellet was kept at -80 �C for up to

4 weeks or immediately processed. Two procedures

were used to lyse cells. For the enzymatic digestion,

100 ll of Tris–EDTA (TE) buffer containing lyso-

zyme (20 mg ml-1) was added to the bacterial pellet,

incubated for 10 min at room temperature and vor-

texed every 2 min. In the second procedure, bacterial

cells were suspended in 100 ll PBS and subjected to 3

freeze–thaw cycles in liquid nitrogen, and subsequent

grounding to a fine powder under liquid nitrogen after

addition of 350 ll of Quiagen buffer RLT. Total RNA

was extracted using an RNeasy Mini kit (Qiagen) with

on-column DNase I digestion following the supplier’s

instructions. A second DNase I treatment was per-

formed in-solution to ensure complete removal of

contaminating DNA, followed by column-based RNA

clean-up (RNeasy Mini kit, Qiagen). The quality of the

isolated RNA was verified by horizontal agarose gel

electrophoresis and RNA quantity was assessed by UV

spectrophotometry.

RT-PCR amplification

The expression of cadF mRNA was analyzed by RT-

PCR using the Promega Access RT-PCR System

according to the manufacturer’s instructions. The

cadF specific primers F2B/R1B were those described

by Konkel et al. (1999). Each RT-PCR reaction was

conducted in a final volume of 25 ll. The reaction

mixture contained 5 ll of 59 buffer, 0.5 ll of dNTPs

mix (10 mM), 1 ll of each primer solution (20 lM),

0.5 ll of enzyme mix and 500 ng of RNA previously

denatured at 65 �C for 10 min. The thermal cycling

profile was as follows: 45 �C for 60 min and 94 �C for

3 min; 45 cycles of 94 �C for 45 s, 45 �C for 1 min,

68 �C for 2 min and a final extension of 68 �C for

7 min. Negative control samples containing sterile

water were always included. DNA contamination was

controlled by performing reactions under identical

conditions in the absence of reverse transcriptase. RT-

PCR products were analyzed by 2 % agarose gel

electrophoresis to check the size of the amplified

fragments by comparison to a DNA molecular weight

marker (BenchTop 100 bp DNA Ladder, Promega,

Milan, Italy).

Generation of the polyclonal CadF antibodies

Polyclonal antiserum (a-CadF-1) was raised by

Biogenes (Berlin, Germany) immunizing two rabbits

with a conserved C. jejuni CadF-derived peptide

(293–306 aa: QDNPRSSNDTKEGR) conjugated to

Limulus polyphemus hemocyanin as carrier protein.

Subsequently rabbit antiserum was purified in order to

obtain immunoglobulin fractions using Protein A

affinity chromatography (Sigma). Antibodies speci-

ficity was increased after indirect co-adsorption on

E. coli (TG-1) bacterial lysate. Western blotting and

immunoblot analysis demonstrate that a-CadF-1 is

highly specific for CadF from C. jejuni (Konkel et al.

1997).

Two dimensional electrophoresis

Bacteria were harvested by centrifugation at 2,000 g

for 10 min. Pellets were resuspended in urea lysis

buffer (8 M urea, 4 % CHAPS, 65 mM DTE, 40 mM

Tris base) and sonicated for 5 s on ice. Insoluble

material was removed by centrifugation at 21,000 g

for 10 min. Protein concentration of samples was

determined by Bradford assay (Bradford 1976). Forty-

five micrograms (analytical runs) or 500 lg (semi-

preparative runs) of proteins were used for each

electrophoretic run. 2-DE was carried out as previ-

ously described (Sestili et al. 2009; Saltarelli et al.

2009). Analytical gels were stained with silver nitrate

(Sinha et al. 2001). Semi-preparative gels for MS

analysis were stained with Brilliant Blue G-Colloidal

(Sigma) according to the manufacturer’s procedure.

Gel images were acquired by Fluor-S MAX multi-

imaging system (BioRad Laboratories, Milan, Italy)

and the data were analysed with ImageMaster 2D

Platinum software. Protein quantification values are

calculated as relative volume (%volume) and relative

Antonie van Leeuwenhoek

123

intensity (%intensity). Gel calibration was carried out

using human plasma as internal standard (Bini et al.

1996).

Immunoblotting

After 2-DE, the separated proteins were transferred to

a nitrocellulose membrane (GE Healthcare). For

immunodetection, a 1:2.000 dilution of the polyclonal

rabbit anti-CadF antibody was incubated overnight at

4 �C. After three 5 min washes, the blot was incubated

for 1 h with the corresponding anti-rabbit HRP-

conjugated secondary antibody (Pierce). Immune

complexes were visualized using the Supersignal

Dura reagent (Pierce).

Protein in-gel digestion and nanoelectrospray

quadrupole time-of-flight tandem mass

spectrometry (nanoESI-Q-TOF MS–MS) analysis

The method for protein in-gel digestion was adapted

from Shevchenko et al. (1996) as previously described

(Guescini et al. 2010). LC–ESI–MS/MS analysis was

performed using a Q-TOF microTM mass spectrometer

(Micromass, Manchester, UK) equipped with a Z-spray

nanoflow electrospray ion source and a CapLC system.

The sample was analyzed using a Symmetry C18

nano column (Waters, Milford, MA, USA) as an

analytical column. For protein identification, MS/MS

spectra were searched by MASCOT (Matrix science,

www.matrixscience.com, UK) using the NCBI nr

database. For unmatched peptides, however, good

quality MS/MS spectra were manually sequenced using

a de novo sequencing process (carried out by PepSeq of

the Masslynx 4.0 software, Micromass) and the

obtained sequence was subsequently used in Expasy

TagIdent.

Epithelial cells

Caco-2 cells, an intestinal cell line derived from a

human colorectal carcinoma that spontaneously dif-

ferentiates under standard culture conditions, were

used for adherence assays. Cells were grown in

Dulbecco’s Modified Eagle’s Medium (D-MEM;

Sigma) supplemented with 10 % fetal bovine serum

(FBS, Pbi, Milan, Italy), 1 % non-essential aminoac-

ids (Sigma) and 1 % antibiotics solution (5,000 U of

streptomycin–penicillin; Sigma) at 37 �C in a 5 %

CO2 humidified atmosphere. For the experimental

assays, Caco-2 cells were seeded at 2 9 104 cell per

well in 6-well plastic plates and incubated for 7 days at

37 �C in a 5 % CO2 humidified atmosphere. Before

the adhesion assay, the cell monolayers were washed

twice with phosphate-buffered saline (PBS) pH 7.2.

Adhesion assay

The adhesion assay was performed as described by

Ganan et al. (2010) with some modifications. Briefly,

20 ml aliquots of each C. jejuni strain from micro-

cosms were aseptically kept at different days of aging

(T0, T7, T21, T35, T46) and centrifuged at 3,500 rpm

for 15 min; the pellets were then resuspended in

D-MEM containing 1 % FBS and 1 ml of this

suspension was inoculated in 6-wells plates containing

semi-confluent Caco-2 cells. The infected monolayers

were incubated for 3 h at 37 �C in 5 % CO2 to allow

bacterial adherence. Cells were washed 3 times with

PBS to remove non-adherent bacteria, lysed with 1 %

Triton X-100 (Sigma) and total bacteria (intracellular

and extracellular bacteria) associated with Caco-2

cells were counted by plating serial dilutions of the

lysates onto Columbia Agar base (Oxoid). The number

of colony forming units (CFU) was assessed after

plates had been incubated for 48 h in microaerophilic

conditions.

Results

Induction of VBNC state

Figure 1 shows the changes in cell numbers of the two

C. jejuni strains during incubation in freshwater

microcosms at 4 �C. Under these conditions, total cell

counts of both strains (about 108 cells/ml) did not

change during the first 7 days incubation in micro-

cosm water. A decrease in cell culturability was

observed after 21 and 35 days, and the VBNC state

was reached after 48 and 46 days of incubation

(\0.1 cfu/ml) for C. jejuni 241 and C. jejuni ATCC

33291, respectively. In the VBNC state *106 cells/ml

that were CTC formazan positive (i.e. metabolically

active) were present in the bacterial population.

Antonie van Leeuwenhoek

123

cadF gene expression in the VBNC state

Until 3 weeks of incubation in microcosms, no

significant differences in total recovery and purity of

isolated RNA for cadF expression were observed

between the two used procedures. After T21, bacterial

cells became increasingly refractory to chemical lysis

as indicated by an extremely reduced or no RNA yield

(data not shown). Therefore, RNA extraction from

microcosm samples after that time was performed

using the mechanical cell lysis procedure.

RT-PCR with CadF-specific primers F2B and R1B

yielded a single amplicon of the expected size (400-

bp) from total RNA with all samples of both C. jejuni

ATCC and C. jejuni 241 strain (Fig. 2). CadF mRNA

was detected during the entire incubation period, but

the signal intensities decreased with increasing loss of

culturability and became weak after transition of the

bacterial populations to non-cultivable state (T46).

When the same samples were subjected to RT-PCR

without reverse transcriptase, no product was detected,

showing that the observed product originated from

reverse-transcribed mRNA and not from residual

chromosomal DNA (data not shown).

CadF protein translation in the VBNC state

To understand if the cadF gene-product is expressed in

C. jejuni strains incubated at 4 �C in freshwater and in

the VBNC state, a proteomic approach was purposed.

1,00E+00

1,00E+01

1,00E+02

1,00E+03

1,00E+04

1,00E+05

1,00E+06

1,00E+07

1,00E+08

1,00E+09

Time (days)

CF

U/m

l

CFU

DAPI

CTC

(a)

(b)

1,00E+00

1,00E+01

1,00E+02

1,00E+03

1,00E+04

1,00E+05

1,00E+06

1,00E+07

1,00E+08

1,00E+09

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Time (days)

CF

U/m

l

CFU

DAPI

CTC

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Fig. 1 Induction of entry into VBNC state of C. jejuni 241

(a) and C. jejuni ATCC 33291 (b) incubated at 4 �C in

freshwater. Culturable cells were counted by the standard plate

count method (cfu/ml) on Columbia agar base; viable and total

cells were enumerated by epifluorescence on CTC-DAPI

staining. Error bars indicate standard deviations

M 1 2 3 4 5 6 7 M M 1 2 3 4 5 6 7 M

(a) (b)

400 bp

Fig. 2 Detection by RT-PCR of cadF mRNA during the entry

to the VBNC state of (a) C. jejuni 241 and (b) C. jejuni ATCC

33291 maintained at 4 �C in freshwater. Amplification was

performed on RNA extracted at different times: lane 1 T0, lane 2

T7, lane 3 T21, lane 4 T35, lane 5 T46, lane 6 positive control

with RNA extracted from a stationary-phase culture of strain

ATCC 33291, lane 7 negative control containing sterile water

Antonie van Leeuwenhoek

123

The 2-DE gel analysis was performed on C. jejuni 241

and C. jejuni ATCC 33291 samples collected at day 0,

25 and in the VBNC state. Two biological replicate

gels with two technical replicates (four gels in total)

were run for each time condition, giving similar results

(Fig. 3). To assess the presence of CadF during

different states of growth, we firstly localized the

protein by immunoblot analysis using a polyclonal

rabbit anti-CadF antibody. The spot corresponding to

the immunoblot signal was excised from the gel, sliced

(a)

(b) (c) (d)

(e) (f) (g)

Fig. 3 Representative 2-DE map of C. jejuni 241 proteins at day 0 (a). Boxed area corresponds to the expanded views of C. jejuni 241

(b–d) and C. jejuni ATCC 33291 (e–g) at day 0 (b, e), 25 (c, f) and in the VBNC state (d, g)

Antonie van Leeuwenhoek

123

into pieces and subsequently subjected to in-gel

digestion with trypsin. The extracted peptides were

then analyzed by nanospray LC–MS/MS, giving the

results showed in Table 1. Our proteomics results

indicate that the adhesion molecule CadF is present at

time 0 and 25 of microcosm incubation and is also

detectable in the VBNC state of both C. jejuni strains,

as showed in the enlarged 2-DE gel parts (Fig. 3).

Notably, the expression level of CadF decreased

during the 3 time points analysed but, although at

low levels, it was also present in the VBNC state as

well. The normalized values of relative intensity

(%intensity) and relative volume (%vol) of the CadF

spot for each time point are given in Table 2.

Adhesion to Caco-2 cells

Regarding the ability of C. jejuni strains to adhere to

cultured Caco-2, the bacterial cells decreased in

adherence efficiency at the different ages of micro-

cosms, compared to the exponential cells. In the

VBNC state, C. jejuni 241 and C. jejuni ATCC 33291

showed 26.9 and 40 % reductions in efficiency of

adherence to Caco-2 cells, respectively, in comparison

with the mid-logarithmic phase cells, for which the

strains showed percentage of adhesion of 52 and 60 %.

The decreasing trends of bacterial adhesion compared

to the culturability and respiratory activity during the

induction of the VBNC state of the C. jejuni strains are

shown in Fig. 4.

Discussion

The pathogenicity of C. jejuni depends mainly on its

ability to adhere and invade the cells of the human

intestine. One of the adhesion factors used by C. jejuni

to attach and eventually invade mammalian cells is

CadF, a binding protein for fibronectin, a component

of the extracellular matrix (Konkel et al. 1997, 2005).

When C. jejuni enters the VBNC state, in response

to environmental stress, it loses culturability, exhibits

enhanced stress resistance, delays mouse lethality and

modifies cell shape and protein profile (Tholozan et al.

1999; Baffone et al. 2006; Zhang et al. 2009). Because

of this last aspect, the aim of this study was to assess the

maintenance of the putative C. jejuni adhesin-encoding

gene cadF, the expression of the related encoded

protein, and to examine changes in cell adhesion in two

C. jejuni strains incubated in freshwater microcosms at

4 �C. In this work, C. jejuni cells became VBNC within

46–48 days. Culturable cell counting confirmed higher

levels of viability compared to culturability of C. jejuni

cells and thus transformation of cells into a VBNC

state. The time needed by both strains to provide non-

culturable forms was longer than in our earlier study

(Baffone et al. 2006), but this was probably due to the

higher inoculum (108 vs 106 cfu/ml) used here to set up

the microcosms. A total RNA extraction procedure and

a reverse-transcriptase assay were developed to

amplify cadF mRNA from C. jejuni cells during

incubation in the freshwater microcosm. Transcripts

Table 1 Identification of CadF protein by LC–ESI–MS/MS

NCBI ID Protein name Nominal mass (Mr) pI Score Coverage (%) Peptides

Q5HSV3_CAMJR Fibronectin-binding protein (CadF) 36,151 5.96 204 12 AVEEVADTR

EGALLDENGCEK

SVANELEK

TVGYGQDNPR

Table 2 The values indicate the mean relative volume (%volume) and mean relative intensity (%intensity) ± mean square deviation

values (N = 4) of the spot corresponding to the CadF protein for C. jejuni 241 and C. jejuni ATCC 33291 strains

Time C. jejuni 241 C. jejuni ATCC 33291

T0 T25 VBNC T0 T25 VBNC

%Intensity (mean) 0.15 ± 0.017 0.10 ± 0.02 0.03 ± 0.009 0.07 ± 0.008 0.04 ± 0.009 0.02 ± 0.009

%Volume (mean) 0.15 ± 0.02 0.05 ± 0.015 0.01 ± 0.009 0.06 ± 0.007 0.02 ± 0.008 0.01 ± 0.008

Antonie van Leeuwenhoek

123

were detected at each time point of sampling, although

the intensity of the signal appeared to decline after

entry into the VBNC state. These results suggest that

CadF mRNA may be constitutively expressed in viable

C. jejuni cells, including non-culturable cells, regard-

less of the origin and serovar of the isolates. It has been

previously demonstrated that VBNC Campylobacter

cells, induced by cold temperature incubation in

nutrient-rich conditions, express the cadF gene

(Chaisowwong et al. 2012). In this study, water was

used as the microcosm since it plays an important role

in the ecology of C. jejuni (Altekruse et al. 1998) and

has been implicated as a vehicle in several outbreaks.

In recent years, considerable evidence has accumu-

lated indicating that virulence factor gene expression is

preserved during the non-culturable state by patho-

genic bacteria, such as cholera toxin genes (ctxAB) in

Vibrio cholerae, a thermostable direct hemolysin gene

(tdh) in Vibrio parahaemolyticus (Vora et al. 2005) and

the virulence factor genes tdh2, escU, vopP and spa24

encoding cytosolic, inner membrane and effector

proteins of type III secretion system 2 TTSS2 in

V. parahaemolyticus (Coutard et al. 2007). The data

obtained by proteomic analysis confirm the results by

RT-PCR, which confirmed expression of this protein

spot. It should be noted that for CadF, as well as the

PEB1 and CDT virulence factors, no significant

differences in their expression were observed between

C. jejuni cultured at 37 and 42 �C both in agar or broth

(Zhang et al. 2009). However, no previous study has

Fig. 4 Culturability,

respiring activity and

adhesion to Caco-2 of

C. jejuni 241 (a) and

C. jejuni ATCC 33291

(b) strains during the

induction of the VBNC state

at 4 �C in freshwater. The

results are presented as

mean ± SD of cfu/ml

Antonie van Leeuwenhoek

123

addressed expression when the microorganism was

preserved under stress conditions such as cold temper-

atures. In our results, proteomic comparison of

C. jejuni cells in both viable and VBNC forms

demonstrated differences in CadF expression between

the two states of growth. This study also showed that

C. jejuni VBNC bacteria retain their ability to adhere to

intestinal epithelial cells, though at a reduced rate, as

do respiring cells. Indeed, there are considerable

discrepancies in the literature concerning the mainte-

nance of the adhesive abilities by VBNC Campylo-

bacter cells. In particular, Cappelier et al. (1999)

showed that VBNC cells, obtained after suspension in

surface water, had lost their adhesion ability, which

was regained after recovery in embryonated eggs.

Verhoeff-Bakkenes et al. (2008) reported that when

INT-407 cells were exposed to culturable C. jejuni with

or without VBNC cells, no differences were found in

the number of bacteria adhering to or invading INT-

407, suggesting that VBNC cells lacked adhesion

properties in vitro. On the other hand, Duffy and Dykes

(2009) demonstrated that C. jejuni cells were able to

attach to stainless steel after they became non-cultur-

able during storage in distilled water at 4 �C for

30 days.

In conclusion, our data show that C. jejuni VBNC

cells express the CadF protein, and, although at

reduced rate, retain their ability to adhere to Caco-2

cells. It could be thus hypothesized that expression of

CadF in the VBNC state may contribute to the

maintenance of the adhesive ability of non-culturable

C. jejuni strains, which may be relevant if these

pathogens were introduced into the animal or human

gut. Indeed, we have previously shown (Baffone et al.

2006) that C. jejuni VBNC forms are able to resus-

citate in a mouse model and the observed results

support this hypothesis. Further investigations are

needed to understand the role of the CadF protein in

the adhesion properties of VBNC cells but our results

provide further evidence supporting the retention of

potential pathogenicity by C. jejuni non-cultivable

forms under stressful environmental conditions.

References

Altekruse SF, Swerdlow DL, Stern NJ (1998) Campylobacterjejuni. Vet Clin North Am Food Anim Pract 14:31–40

Baffone W, Casaroli A, Citterio B, Pierfelici L, Campana R,

Vittoria E, Guaglianone E, Donelli G (2006) Campylo-bacter jejuni loss of culturability in aqueous microcosms

and ability to resuscitate in a mouse model. Int J Food

Microbiol 107:83–91

Barer MR, Harwood CR (1999) Bacterial viability and cultu-

rability. Adv Microb Physiol 41:93–137

Bini L, Sanchez-Campillo M, Cantucci A, Magi B, Marzocchi

B, Comanducci M, Christiansen G, Birkelund S, Cevenini

R, Vretou E, Ratti G, Pallini V (1996) Mapping of Chla-mydia trachomatis proteins by immobiline-polyacrylamide

two-dimensional electrophoresis: spot identification by

N-terminal sequencing and immunoblotting. Electropho-

resis 17:185–190

Bradford M (1976) Rapid and sensitive method for the quantita-

tion of microgram quantities of protein utilizing the principle

of protein-dye binding. Anal Biochem 72:248–254

Butzler JP (2004) Campylobacter, from obscurity to celebrity.

Clin Microbiol Infect 10:868–876

Cappelier JM, Lazaro B, Rossero A, Fernandez-Astorga A,

Federighi M (1997) Double staining (CTC-DAPI) for

detection and enumeration of viable but non-culturable

Campylobacter jejuni cells. Vet Res 28:547–555

Cappelier JM, Minet J, Magras C, Colwell RR, Federighi M

(1999) Recovery in embryonated eggs of viable but non-

culturable Campylobacter jejuni cells and maintenance of

ability to adhere to HeLa cells after resuscitation. Appl

Environ Microbiol 65:5154–5157

Chaisowwong W, Kusumoto A, Hashimoto M, Harada T,

Maklon K, Kawamoto K (2012) Physiological character-

ization of Campylobacter jejuni under cold stresses con-

ditions: its potential for public threat. J Vet Med Sci

74(1):43–50

Colwell RR, Huq A (1994) Environmental reservoir of Vibriocholerae. The causative agent of cholera. Ann NY Acad

Sci 740:44–54

Coutard F, Lozach S, Pommepuy M, Hervio-Heath D (2007)

Real-Time Reverse Transcription-PCR for transcriptional

expression analysis of virulence and housekeeping genes in

viable but nonculturable Vibrio parahaemolyticus after

recovery of culturability. Appl Environ Microbiol

73:5183–5189

Dorrell N, Wren BW (2007) The second century of Campylo-bacter research: recent advances, new opportunities and

old problems. Curr Opin Infect Dis 20:514–518

Duffy LL, Dykes GA (2009) The ability of Campylobacter je-juni cells to attach to stainless steel does not change as they

become nonculturable. Foodborne Pathog Dis 6:631–634

Ganan M, Campos G, Munoz R, Carrascosa AV, de Pascual-

Teresa S, Martinez-Rodriguez AJ (2010) Effect of growth

phase on the adherence to and invasion of Caco-2 epithelial

cells by Campylobacter. Int J Food Microbiol 140:14–18

Guescini M, Guidolin D, Vallorani L, Casadei L, Gioacchini

AM, Tibollo P, Battistelli M, Falcieri E, Battistin L, Agnati

LF, Stocchi V (2010) C2C12 myoblasts release micro-

vesicles containing mtDNA and proteins involved in signal

transduction. Exp Cell Res 316:1977–1984

Ketley JM (1997) Pathogenesis of enteric infection by Cam-pylobacter. Microbiology 143:5–21

Konkel ME, Garvis SG, Tipton SL, Anderson DE Jr, Cieplak W

Jr (1997) Identification and molecular cloning of a gene

Antonie van Leeuwenhoek

123

encoding a fibronectin-binding protein (CadF) from Cam-pylobacter jejuni. Mol Microbiol 24:953–963

Konkel ME, Gray SA, Kim BJ, Garvis SG, Yoon J (1999)

Identification of the enteropathogens Campylobacter jejuniand Campylobacter coli based on the cadF virulence gene

and its product. J Clin Microbiol 37:510–517

Konkel ME, Christensen JE, Keech AM, Monteville MR, Klena

JD, Garvis SG (2005) Identification of a fibronectin-bind-

ing domain within the Campylobacter jejuni CadF protein.

Mol Microbiol 57:1022–1035

Korhonen LK, Martikainen PJ (1991) Comparison of the survival of

Campylobacter jejuni and Campylobacter coli in culturable

form in surface water. Can J Microbiol 37:530–533

Ma Y, Hanning I, Slavik M (2009) Stress-induced adaptive

tolerance response and virulence gene expression in

Campylobacter jejuni. J Food Safety 29:126–143

Monteville MR, Yoon JE, Konkel ME (2003) Maximal adher-

ence and invasion of INT 407 cells by Campylobacter je-juni requires the CadF outer-membrane protein and

microfilament reorganization. Microbiology 149:153–165

Oliver JD (1993) Formation of viable but nonculturable cells. In:

Kjelleberg S (ed) Starvation in bacteria. Plenum Press,

New York, pp 239–272

Pruzzo C, Tarsi R, Lleo MM, Signoretto C, Zampini M, Colwell

RR, Canepari P (2002) In vitro adhesion to human cells by

viable but nonculturable Enterococcus faecalis. Curr

Microbiol 45:105–110

Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF (1992) Use of

a fluorescent redox probe for direct visualisation of actively

respiring bacteria. Appl Environ Microbiol 58:1801–1808

Rollins DM, Colwell RR (1986) Viable but nonculturable stage of

Campylobacter jejuni and its role in survival in the natural

aquatic environment. Appl Environ Microbiol 52:531–538

Saltarelli R, Ceccaroli P, Iotti M, Zambonelli A, Casadei L,

Vallorani L, Stocchi V (2009) Biochemical characteriza-

tion and antioxidant activity of mycelium of Ganodermalucidum from Central Italy. Food Chem 116:143–151

Sestili P, Barbieri E, Martinelli C, Battistelli M, Guescini M,

Vallorani L, Casadei L, D’emilio A, Falcieri E, Piccoli G,

Agostani D, Annibalini G, Paolillo M, Gioacchini AM,

Stocchi V (2009) Creatine supplementation prevents the

inhibition of myogenic differentiation in oxidatively

injured C2C12 murine myoblasts. Mol Nutr Food Res

53:1187–1204

Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass

spectrometric sequencing of proteins silver-stained poly-

acrylamide gels. Anal Chem 68:850–858

Sinha P, Poland J, Schnolzer M, Rabilloud T (2001) A new

silver staining apparatus and procedure for matrix-assisted

laser desorption/ionization-time of flight analysis of pro-

teins after two-dimensional electrophoresis. Proteomics

1:835–840

Snelling WJ, Matsuda M, Moore JE, Dooley JS (2005) Cam-pylobacter jejuni. Lett Appl Microbiol 41:297–302

Tholozan JL, Cappelier JM, Tissier JP, Delattre G, Federighi M

(1999) Physiological characterization of viable-but-non-

culturable Campylobacter jejuni cells. Appl Environ

Microbiol 65:1110–1116

Verhoeff-Bakkenes L, Hazeleger WC, Zwietering MH, De

Jonge R (2008) Lack of response of INT-407 cells to the

presence of non-culturable Campylobacter jejuni. Epi-

demiol Infect 136:1401–1406

Vora GJ, Meador CE, Bird MM, Bopp CA, Andreadis JD,

Stenger DA (2005) Microarray-based detection of genetic

heterogeneity, antimicrobial resistance, and the viable but

nonculturable state in human pathogenic Vibrio spp. Proc

Natl Acad Sci USA 102:19109–19114

Young KT, Davis LM, Dirita VJ (2007) Campylobacter jejuni:molecular biology and pathogenesis. Nat Rev Microbiol

5:665–679

Zhang MJ, Xiao D, Zhao F, Gu YX, Meng FL, He LH, Ma GY,

Zhang JZ (2009) Comparative proteomic analysis of

Campylobacter jejuni cultured at 37�C and 42�C. Jpn J

Infect Dis 62:356–361

Antonie van Leeuwenhoek

123