Journal of Microbiological Methods 83 (2010) 17

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Journal of Microbiological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

A method for the growth and recovery of 17 species of Campylobacter and its
subsequent application to inoculated beef
.A. Lynch a, C. Cagney a, D.A. McDowell b, G. Duffy a,
a
Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland
b
University of Ulster, Jordanstown campus, Shore road, Newtownabbey, Co. Antrim,BT37 OQB, Northern Ireland, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to develop a universal cultural protocol, which could facilitate the growth of 17
Received 11 March 2010 species and 3 subspecies of Campylobacter. Enrichment media including Campylobacter Enrichment Broth
Received in revised form 28 May 2010 (CEB) and Bolton Broth were tested against a panel of Campylobacter strains (n = 53) encompassing 17 species
Accepted 7 June 2010
and 3 subspecies, under a gas atmosphere containing hydrogen (2.5% O2, 7% H2, 10% CO2, and 80.5% N2). The
Available online 11 June 2010
impact of enrichment conditions on cell motility was also investigated using uorescent microscopy.
Membrane ltration was examined as a means of selectively recovering Campylobacter from enrichment media
Keywords:
Campylobacter
on two different non-selective agars, Anaerobe Basal Agar (ABA) and Tryptose Blood Agar (TBA). The results
Emerging Campylobacter culture media showed that enrichment in CEB for 24 h at 37 C under a modied gas atmosphere followed by centrifugation
Membrane ltration and membrane ltration onto ABA allowed recovery of all species (53 strains) of Campylobacter from
Morphology inoculated meat samples. After 24 h enrichment, there were higher levels of motile Campylobacter in CEB than
Motility in Bolton broth and it is proposed that this attribute aided the passage of the Campylobacter through the
Isolation membrane lter. The results of this study provide a simple, but effective method for the growth and recovery of
Beef a wide range of diverse Campylobacter spp. from a meat matrix using common cultural parameters.
2010 Elsevier B.V. All rights reserved.

1. Introduction culturally fastidious Campylobacter species are also frequently linked

Following its recognition as a major cause of diarrhoeal illness
(Middlekamp and Wolf, 1961), the Campylobacter genus has become
the focus of considerable research. Taxonomically, the Campylobac-
teraceae family is still evolving, partly due to its cultural fastidious-
ness, with 24 species (6 subspecies) of Campylobacter now described.
Campylobacter jejuni and Campylobacter coli are largely identied as
the predominate cause of Campylobacter-related gastric-illness (Kuusi
et al., 2004; Jimenez et al., 2005; Mazick et al., 2005), and have
therefore received the greatest attention in terms of research.
Although cases of campylobacteriosis are generally self-limiting
(Butzler, 1982), they can lead to more serious sequale such as
Miller-Fisher and GuillanBarr syndromes (Rees et al., 1995; Sallo-
way et al., 1996; Nachamkin et al., 2000), however such instances are
rare.
More recently, species other than C. jejuni and C. coli including,
Campylobacter concisus, Campylobacter hyointestinalis, Campylobacter
upsaliensis, Campylobacter gracilis and Campylobacter curvus have
been reported to cause diarrhoeal illness in humans (Lindblom et al.,
1995; Gorkiewicz et al., 2002; Labarca et al., 2002; Lastovica and Le
Roux, 2003; Maher et al., 2003; Boyanova et al., 2004). These
Corresponding author. Tel.: + 353 1 8059500; fax: + 353 1 8059550.
E-mail address: geraldine.duffy@teagasc.ie (G. Duffy).
to serious extra-intestinal illnesses such as joint and organ infections,
cardiac problems and meningitis (Bingham et al., 1992; Gaudreau and
Lamothe, 1992; Morooka et al., 1992; Dronda et al., 1998; Martinet
et al., 2001; Herve et al., 2004; Han et al., 2005; Ahmar et al., 2008;
Hull and Varma, 2009).
A number of factors have hindered signicant research into these
emerging species, the most fundamental of which is the lack of a
suitable universal cultural system. Current routine isolation condi-
tions involve incubation in a thermophilic, microaerobic environment
of 510% C02 at 41.5 C (Hunt et al., 1998; Anonymous, 2006).
Although optimal for the growth of C. jejuni and C. coli, these methods
create a bias, reducing the probability of recovering other Campylo-
bacter spp., including the anaerobes and non-thermophiles (Carlone
and Lascelles, 1982; Steele et al., 1985; Humphrey, 1986; Vandamme,
2000). A number of clinically signicant species require a hydrogen-
rich atmosphere (Lawson et al., 2001) and/or extended incubation
periods of several hours to a number of weeks (On et al., 1996; Lawson
et al., 2001). Most cultural media also contain antibiotics that have
been shown to prohibit the growth of many species (Goossens et al.,
1986; Loades et al., 2006), leading to increased selection of C. jejuni
and C. coli over other less tolerant species. Previously, in order to
prevent the inhibition of these culturally diverse species, researchers
had to rely upon several different growth and isolation techniques
(Misawa et al, 2000; Schroeder et al., 2003; Uzunovic-Kamberovie
et al, 2007; Vereen et al., 2007). The culmination of these problems

0167-7012/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2010.06.003
2 .A. Lynch et al. / Journal of Microbiological Methods 83 (2010) 17

has meant that screening for non-C. jejuni/C. coli species is a labour- Table 1
intensive, lengthy and costly procedure, one which may not Collection of Campylobacter used in study.

economically feasible for many diagnostic laboratories. Consequently, Species Laboratory sources
these rarer, yet potentially signicant species are overlooked during
DIFVRa University of
clinical testing. Cape Townb
A method reported by Le Roux and Lastovica (1998) enables the
C. coli RM 2228, Lab 33 300.97
recovery of a wider variety of Campylobacter species, by avoiding the C. concisus CCUG 13144, CCUG 19995 Lastovica 396-96
commonly accepted isolation procedures and media. The method, C. curvus SSI 19296 Lastovica 525.92,
known as the Cape Town Protocol exploits the natural motility of Lastovica 13A
these pathogens, by incorporating a membrane lter which acts as a C. fetus subsp. fetus CCUG 32114, Abdn 1076 241.99
C. fetus subsp. CCUG 11287, Armi 4402
selective barrier against non-motile and larger motile competing venerealis
organisms, effectively eliminating the need for chemical selective Abdn SM5
agents, and reducing the risks of suppressing the growth and recovery C. gracilis CCYG 27720, CCUG 13143
of some Campylobacter spp. To this end, a basal agar (antibiotic-free) C. helveticus CCUG 30566, CCUG 34016
C. hominis NCTC CHOO1, NCTC CHOO3
can be used. Using this approach C. upsaliensis and C. concisus were
C. hyointestinalis subsp. LMG 7538, LMG 9260 179.96, 234.95
found to be present in almost 50% of diarrhoeal stool samples hyointestinalis
examined at the Red Cross Children's hospital in Cape Town (Le Roux C hyointestinalis subsp. CHY 5, CCUG 27631
and Lastovica, 1998). The method was recently used in geographically lawsonii
independent clinical studies (Alam et al., 2006; McNulty et al., 2008) C. jejuni subsp. doylei SSI 5384, CCUG 18266 269.97
C. jejuni subsp. jejuni NCTC 11168, RM 1221 47.97
and the results mirrored those seen in the South African study. SVS 4039, RM 1864
To date, the protocol has been used exclusively for clinical (= 81176)
specimens of symptomatic patients carrying a high bacterial load, C. lanienae NCTC 13004, DARDNI G718D
therefore in its current format the method does not lend itself to the C. lari RM 2100 (NARTC), CCUG 22395
C. mucosalis CCUG 21559, CCUG 23201
examination of food samples in which the organism may be injured or
C. rectus CCUG 20446, CCUG 11645
present in low numbers. The objective of this study was to the C. showae CCUG 30254, CCUG 11641
establish enrichment, recovery and detection methods suitable for the C. sputorum LMG 11764, CCUG 20703 Lastovica 86.92
cultivation of a wide range of diverse Campylobacter species. The C. upsaliensis CCUG 19559, CCUG 19607
developed methods were used to adapt the Cape Town Protocol to C. ureolyticus CCUG 18470, Rigshospitalet 9880

facilitate the potential recovery of these species from a meat matrix a

Danish Institute for food and Veterinary Research, Department of Microbiological
using articially inoculated beef. Food Safety, Bulowsvej 27, DK-1790 Copenhagen, Denmark.
b
Department of Medical Microbiology, Medical school, University of Cape Town,
Observatory, 7925, Cape Town, South Africa.
2. Materials and methods

supplied with the previously described gas mixture for 24 and 48 h at

2.1. Bacterial strains
The Campylobacter strains used in this study (n = 53) were
supplied by The Danish Institute for Food and Veterinary Research
(Copenhagen, Denmark), and The University of Cape Town (South
Africa), through the EU Framework 5 Campycheck Project (QLK1 CT
2002 02201) (Table 1). All strains were maintained on Protect beads
(Technical Services Consultants Ltd, Lancashire, U.K), according to
manufacturer instructions and stored at 80 C.
2.2. Preparation of bacterial inoculum
Hunts broth (30 ml) (Hunt et al., 1998) with 5% lysed horse blood
(Oxoid, Wade Road, Basingstoke, U.K) and ferrous sulfate, sodium
metabisulfate and sodium pyruvate supplement (FBP) (George et al.,
1978), was inoculated with a single protect bead of each strain for
resuscitation. Cultures were incubated under the gas atmosphere 2.5%
O2, 7% H2, 10% CO2, and 80.5% N2 (Keevil et al., 2006) at 37 C for 48 h in a
compact anaerobic work station (Don Whitley, West Yorkshire, U.K).
After incubation, each strain was diluted 1:100 in Hunts broth and
incubated for an additional 24 h. The cultures were centrifuged at 29 g
(5000 rpm) for 10 min at 4 C and the resulting pellet was washed and
resuspended three times in 9 ml Maximum Recovery Diluent (MRD)
(Oxoid) at 29 g (5000 rpm) for 4 min at 4 C.
2.3. Enrichment
The growth of Campylobacter strains (n= 53) in differing enrich-
ment and plating media was assessed. Inocula were prepared as
described previously, serially diluted in MRD and added to either
Campylobacter enrichment broth (CEB) (Lab M, Lancashire, U.K.) or
Bolton Broth (Oxoid) with 5% lysed horse blood to nal concentrations
of 1.5 Log10 CFU ml1. Inoculated broths were incubated in a gas cabinet
37 C. After incubation, strains were serially diluted in MRD and 0.1 ml
of each broth was plated onto Tryptose Blood Agar (TBA) (Oxoid) and
Anaerobe Basal Agar (ABA) (Oxoid), each with 5% lysed horse blood.
Agar plates were dried in a laminar air ow hood for 20 min prior to use
(Nuare Biological cabinets, Davidson and Hardy, Ireland). The inocula
were evenly distributed over the agar surface using a sterile spreader
(Sarstedt, Wexford, Ireland). Incubation conditions were as described
previously. After 48 h, plates were examined and the numbers of
recovered Campylobacter (log10 CFU ml1) were estimated.
2.4. Motility
The motility of Campylobacter strains grown in the above enrichment
broths (CEB and Bolton) was assessed using NanoOrange (Molecular
Probes, The Netherlands) staining by the method of Grossart et al.
(2000) and Alonso et al. (2002). While stable in aqueous solutions,
NanoOrange dye uoresces when bound to the hydrophobic region of
proteins (Handbook of molecular probes and research chemicals,
Molecular probes; Alonso et al., 2002). Following 24 h incubation,
10 l bacterial suspension and 0.5 l NanoOrange dye was transferred to
the well of a concavity slide. A cover slip was applied and slides were
kept in a dark environment for 15 min to allow complete binding of the
dye. Slides were viewed under 10 and 100 magnications using
Epiuorescent microscopy (Olympus BX26, Olympus, Japan) (Excitation
wavelength 485 nm; emission wavelength 509 nm). On average, 10
elds of vision were examined per slide and samples displaying the
motility typical of Campylobacter were recorded as motile.
2.5. Membrane ltration
The ability of Campylobacter cells to pass though membrane lters
composed of differing materials and pore sizes was assessed.
 .A. Lynch et al. / Journal of Microbiological Methods 83 (2010) 17 3

C. concisus (CCUG 13144) was chosen as a representative of a previously described conditions. Typical Campylobacter colonies were
fastidious hydrogen-requiring clinically signicant emerging species counted and conrmed using the KOH and O.B.I.S tests. Recovered
and C. jejuni (NCTC 11168) was chosen to represent a classic isolates were further conrmed using the PCR method of Linton et al.,
microaerobic species. Inocula were prepared as described above and 1996 for the identication of the genus Campylobacter. C. jejuni, C. coli,
serially diluted in MRD to a level of approximately 2 log10 CFU ml1. C. lari, C. upsaliensis, C. helveticus, C. sputorum, C. fetus, C. curvus,
Cellulose nitrate, cellulose acetate, polycarbonate and mixed ester C. mucosalis and C. concisus were conrmed by PCR (Bastyns et al., 1995;
membrane lters (Whatman, United Kingdom) of various pore sizes Klena et al., 2004; Personal communication, Miller, W. G, Parker, C. T and
(0.45 and 0.6 m) were aseptically placed on the surface of a TBA Mandrell, R.E). The remaining species were conrmed according to the
plate. An aliquot of 0.5 ml of the prepared inocula was transferred in biochemical matrix outlined in the original Cape Town Protocol (Le
droplets to the surface of each test membrane. After 15 min the Roux and Lastovica, 1998). The experiment was repeated in triplicate.
membranes were carefully removed using a sterile tweezers, ensuring
no spillage of residual bacterial suspensions occurred. The ltrate was 2.8. Statistical analysis
spread evenly over the agar surface using a sterile disposable
inoculum spreader and the plates incubated at 37 C for 48 h in gas Three replicate experiments were carried out for all methods
jars with microaerobic gas generation sachets (Oxoid). investigated in this study. Analysis of variance (ANOVA) was carried
out at the 0.001 level of signicance using Genstat 9.1 (Rothamsted
2.6. Recovery of Campylobacter from meat Experimental Station, Harpenden, United Kingdom) to determine if
the growth of various Campylobacter species differed signicantly
The surface of rump beef pieces (25 g) were inoculated with between enrichment methods used. Data generated on the motility of
C. concisus CCUG 19995 by immersing the meat for 60 s in a 100 ml strains in the various enrichment broths was analysed using Pearson's
aliquot of approximately 2 log10 CFU ml1 suspension. The inoculated chi-square to establish if enrichment media had an effect on cell
meat was then hung in a laminar air ow hood for 9 min to dry. motility.
Previous studies (unpublished data) had shown that this approach
allowed for the greatest attachment of Campylobacter to the meat 3. Results
matrix. The inoculated meat was transferred to a lter stomacher bag
(Blender bag, model 400, Grade Packaging Ltd, Leicestershire, U.K) Table 2 shows the number of Campylobacter recovered on two
with 225 ml CEB and 5% lysed horse blood and homogenised in a different agars (log10 CFU ml1) TBA and ABA, following 24 h
pulsier for 15 s (Pulsier, Microgen Bioproducts, UK). A Pulsier enrichment in either CEB or Bolton Broth at 37 C. All 53 strains,
is a vibrated mixer, which can be used as an alternative to the representing 17 species and 3 subspecies of Campylobacter grew well
conventional stomacher (ISO:7218:2007(E)). A vibrating platform in both enrichment broths reaching populations ranging from 5.95
produces high frequency shock waves, which release the microbes Log10 CFU ml1 to 7.86 Log10 CFU ml1 in Bolton broth and 68 Log10
from the food sample. In addition to a faster run time, pulsifying is less CFU ml1 in CEB in the 24 h enrichment period. C. jejuni subsp. jejuni,
destructive than stomaching, producing lower concentrations of C. lanienae, C. lari, C. mucosalis, C. rectus, C. showae, C sputorum and
debris (Kang and Dougherty, 2001). The samples were enriched for C. upsaliensis reached higher levels (P b 0.001) in CEB compared to
24 h at 37 C in a gas cabinet supplied with the previously described Bolton broth, in which only C. concisus reached signicantly higher
gas mixture. Duplicate aliquots (10 ml) of enriched samples were
removed, one of which was subjected to centrifugation at 7 g
Table 2
(2500 rpm) for 15 s. Volumes of 0.2, 0.3, 0.4 and 0.5 ml were removed Mean number of Campylobacter (log10 CFU ml1) under a gas atmosphere of 2.5% 02, 7%
from the centrifuged (supernatant) and non-centrifuged samples, and H2, 10% C02 and balance N2 at 37 C in various enrichment/plating schemas.
transferred to a mixed ester membrane (pore size 0.65 m) on the
Species No. of Mean count (Log10 CFU ml1)
surface of a TBA plate in droplets. After 15 min, membranes were strains
carefully removed, and the inoculum was spread evenly over the Bolton brotha CEBa
included
surface of the agar using sterile inoculum spreaders. All plates were TBAb ABAc TBAd ABAe
incubated in a gas cabinet supplied with the previously described gas C. coli 3 6.92 7.17 6.97 6.57
mix for 48 h at 37 C. Typical Campylobacter colonies were counted C. concisus 3 6.9 7.86sd 6.62 6.46
and conrmed using the KOH test for Gram reaction (Halebian et al., C. curvus 3 6.82 6.81 6.62 6.48
1981). The O.B.I.S Campylobacter test (Oxoid) was then used to test for C. fetus subsp. fetus 3 6.73 6.87 6.21 6.45
C. fetus subsp. venerealis 3 6.6 6.81 6.32 6.6
the presence of L-alanine aminopeptidase (L-ala) in gram-negative C. gracilis 2 6.73 6.7 6.66 6.6
bacteria. As Campylobacteraceae are uniquely KOH positive and L-ala C. helveticus 2 7.12 6.81 6.55 7.27
negative (Carlone et al., 1982; Smith et al., 2006), the combination of C. hominis 2 6.74 6.48 6.29 6.5
both tests provides a rapid and convenient method for differentiating C. hyointestinalis subsp. 4 6.9 6.73 7.08 6.55
hyointestinalis
between Campylobacter and other competing bacteria.
C. hyointestinalis subsp. 2 6.96 6.92 6.59 6.49
lawsonii
2.7. Application of method to meat C. jejuni subsp. doylei 3 6.86 7.08sd 6.55 6.59
C. jejuni subsp. jejuni 5 6.51 6.58 6.44 8sd
Rump beef pieces (25 g) (n= 53) were inoculated with Campylo- C. lanienae 2 6.51 6.85 6.73 8sd
C. lari 2 6.79 6.7 6.64 7.3sd
bacter strains (n= 53, Table 1) to a level of 1.5 log10 CFU cm2 using the
C. mucosalis 2 6.34 6.47 6.58 7.29sd
above described protocol. The samples were placed in a lter stomacher C. rectus 2 6.34 6.8 6 8sd
bag with 225 ml CEB and 5% lysed horse blood and pulsied for 15 s. C. showae 2 6.38 6.46 6.58 8sd
Samples were enriched overnight under previously described incuba- C. sputorum 3 7.09 6.34 6.52 7.27sd
C. upsaliensis 3 6.8 6.56 6.37 7.47sd
tion conditions. After enrichment, 10 ml aliquots of each sample were
C. ureolyticus 2 6.78 6.41 6.52 6.58
removed and centrifuged at 7 g (2500 rpm) for 15 s. From the upper
part of the supernatant, 0.2 ml of each was removed and transferred to a Standard error in the difference (SED) of means of counts (log10 CFU ml1) achieved
using differing enrichment broths a0.099.
mixed ester membrane on the surface of an ABA plate in droplets. After Standard error in the difference (SED) of means (log10 CFU ml1) between species
15 min, the lters were removed and the inoculum was spread evenly under various enrichment/plating schemas: b 0.051, c 0.047, d 0.053, e 0.049.
over the agar surface. Plates were incubated for 48 h under the sd
Signicant difference (p b 0.001).
4 .A. Lynch et al. / Journal of Microbiological Methods 83 (2010) 17

levels (P b 0.001). Overall, the use of ABA plates yielded signicantly CFU g2 (Table 4). It was noted that there were interspecies
(P b 0.001) higher counts for 10 of the 17 species tested and the differences in colony morphologies ranging from the typical grey
highest recovery was achieved when ABA was used in conjunction appearance of C. jejuni to small translucent colonies with a slightly
with CEB. Extending the enrichment period from 24 to 48 h yielded no pink hue for C. concisus. The use of the O.B.I.S kit combined with the
signicant increase in number of Campylobacter recovered and the KOH test gave good presumptive identication of colonies and there
trends noted for species mirrored those seen at 24 h (results not was full agreement between these results and the genus-specic PCR.
shown).
The motility of Campylobacter following the 24 h enrichment 4. Discussion
period in Bolton or CEB is shown in Table 3. Chi-square analysis
showed that motility of strains was signicantly better in CEB than for The recovery and isolation of Campylobacter species other than
strains grown in Bolton broth (P b 0.001). Following 24 h enrichment C. jejuni and C. coli from both clinical and food samples has been
in Bolton broth, only 7 of the 20 species/subspecies tested exhibited hampered by the continuing use of cultural conditions which actively
active motility in the majority of cells observed per eld of vision suppress the growth of the lesser-known species. This study reports a
compared with 17 of 20 species/subspecies enriched in CEB. No method, which enabled the growth and recovery of 17 species and 3
signicant improvement in motility was observed when enrichment subspecies of Campylobacter using a common cultural protocol.
time was increased to 48 h for either of the enrichment broths tested. Although previous studies have attempted to adapt the growth
The recovery of Campylobacter strains (n = 2) was poor following atmosphere, the hydrogenoxygen ratio used, failed to facilitate the
passage of the inocula through cellulose acetate and cellulose nitrate growth of hydrogen-requiring species (Inglis et al, 2010). The
membranes with a pore size of 0.45 m particularly for C. concisus, atmosphere used in this study incorporated a lower level of oxygen
recovering only 30% and 15% of the inocula respectively. Cellulose (2.5%) with 7% hydrogen, which may stimulate the use of alternative
nitrate membranes with a larger pore size of 0.6 m increased the electron pathways in the organisms (Carlone and Lascelles, 1982;
recovery to 100% of C. jejuni and 75% of C. concisus inocula. The use of Gillesepie and Barton, 1996; Sellars et al., 2002; Laanbroek et al., 2004;
polycarbonate membranes (0.6 m) resulted in the formation of a Pittman and Kelly, 2005; Keevil et al., 2006). Despite an optimum
lawn, or conuent growth, with no distinct colony formations. This growth temperature of 41.5 C, incubating at this temperature can
phenomenon was noted with each replicate of the experiment. Mixed lead to oxidative stress in certain Campylobacter strains (Garenaux
ester membranes (0.65 m) facilitated recovery of 100% of the et al., 2008). The growth rate of C. jejuni has been shown to be higher
inoculum for both test species. at 37 C (Khanna et al., 2006). This lower incubation temperature also
The results demonstrated that lightly centrifuging (7 g for 10 s) the improves the recovery of damaged cells (Humphrey, 1986) and can
enriched samples prior to ltration led to an average increase in counts reduce the number of false negative results from food samples (Scates
of 1.5 log10 CFU ml1 over the non-centrifuged samples. The impact of et al., 2003).
volume of aliquot transferred was also examined. The results indicated Despite being produced using the same formula, enrichment in
no signicant difference in recovery between 0.2 ml and 0.5 ml, with CEB yielded higher populations of a number of species compared with
counts ranging between 6.67 and 6.89 log10 CFU ml1. However, the Bolton broth. Such variations in the performance of the enrichment
use of volumes greater than 0.3 ml resulted in the accumulation of media could be attributed to differing peptone sources, which has
excess liquid on the membrane surface, increasing the likelihood of previously been shown to affect bacterial growth and survival
spillage and therefore contamination of the agar surface by competing signicantly (De Speigeleer et al., 2004; Gray et al., 2008).This is not
microora, during membrane removal. the rst report of seemingly identical media displaying considerably
When the developed method (Fig. 1) was applied to meat samples, different recovery rates from pure cultures (Baylis et al., 2000).
each inoculated with a single strain of Campylobacter; all 53 strains NanoOrange was used to examine the motility of cells in a cavity
were successfully recovered to a range of between 7.9 and 8.23 log10 slide. Cells grown in CEB showed higher levels of motility, compared
with those enriched in Bolton broth. Increasing the enrichment time
to 48 h (results not shown) yielded no statistically signicant increase
Table 3
Motility of Campylobacter strains following 24 h enrichment in Campylobacter in cell concentration or motility, indicating that 24 h enrichment in
Enrichment broth (CEB) (5% lysed horse blood) or Bolton broth (5% lysed horse CEB is more compatible with the use of a motility-based ltration
blood) at 37 C under atmosphere (2.5% O2, 7% H2, 10% CO2, and 80.5% N2). method. This is in keeping with a previous study, which showed that
Species No. strains Bolton broth CEBsd Campylobacter motility was at its fastest following 24 h growth in
included liquid media (Karim et al., 1998). Although previously used to
C. coli 3 Motile Motile
examine other agellated bacteria (Grossart et al., 2000), in this study,
C. concisus 3 Motile Motile the authors found NanoOrange to be inconsistent in staining the
C. curvus 3 Motile Motile agella of Campylobacter cells. This failure to uoresce cellular agella
C. fetus subsp. fetus 3 Motile has been reported in another recent study (Heese and Kim, 2009).
C. fetus subsp. venerealis 3 Motile
Both solid media examined facilitated growth of all test strains of
C. gracilis 2 Motile Motile
C. helveticus 2 Motile Motile Campylobacter, however; statistically, ABA gave higher populations
C. hominis 2 than TBA (P b 0.001) for 10 of the species examined. Of these 10
C .hyointestinalis subsp. hyointestinalis 4 Motile Motile species only one, C. lanienae, has not been implicated in human
C. hyointestinalis subsp. lawsonii 2 Motile infection. Colonies formed on ABA were also larger than those on TBA,
C. jejuni subsp. doylei 3 Motile
which could aid in the identication of Campylobacter from mixed
C. jejuni subsp. jejuni 5 Motile Motile
C. lanienae 2 Motile cultures. ABA has a far more complex formula than TBA. Haemin and
C. lari 2 Motile Ferric pyrophosphate provide iron (Parkhill et al., 2000; Genco and
C. mucosalis 2 Motile Dixon, 2001), which is essential in the survival and growth of
C. rectus 2 Motile
Campylobacter (Woodward and Williams, 1993). The inclusion of
C. showae 2 Motile
C. sputorum 3 starch and sodium pyruvate in the recipe protects against toxic
C. upsaliensis 3 Motile metabolites and oxygen derivatives (Smibert, 1978; Bolton et al.,
C. ureolyticus 2 Motile 1984; Stead and Park, 2000). ABA also contains the anaerobe growth
: Denotes lack of motility. promoter, cysteine (Shanson and Singh, 1981). By comparison, TBA,
sd
Signicant difference (p b 0.001). used in the original Cape Town protocol, has a far more basic formula
 .A. Lynch et al. / Journal of Microbiological Methods 83 (2010) 17 5

Fig. 1. Flow diagram of protocol for the recovery of emerging Campylobacter spp. from meat.

containing merely 3 components, Lab-lemco powder, NaCl and membrane, potentially reducing selection based on motility. Negligi-
Tryptose in its basal form. ble adsorption of liquid by the polycarbonate lter (Anonymous,
This study investigated the use of a range of lters, which were 2007) can results in excess uid passing through the membrane and
chosen based on pore size (0.45 and 0.6 m) and matrix composition. onto the agar surface. As Campylobacter has a tendency to swarm and
Results conrmed that the recovery of Campylobacter cells was form a lawn of growth on wet agar (Buck and Kelly, 1981; Shane and
dependant on both pore size and membrane material. The best Stern, 2003), this would account for the conuent growth observed
recoveries were obtained using a 0.65 m mixed cellulose ester for each experimental replicate using polycarbonate membranes. The
membrane. This result was corroborated in the recently published lattice formation of the mixed ester membranes adsorbs liquid over a
study by Speegle et al., 2009. Mixed cellulose ester membranes are large internal surface area, and would explain the clearer colony
composed of cellulose acetate (20%) and cellulose nitrate (80%) with morphology seen with mixed ester over polycarbonate membranes in
pores in a lattice formation, whereas, polycarbonate membranes have this study.
cylindrical pores that allow passive transport of bacteria though the When the above basic enrichment-membrane ltration system
was applied to inoculated beef pieces, it was found that the level of
particulate material in the enriched sample reduced the passage of
Table 4
Mean number of Campylobacter (log10 CFU g2) recovered from the inoculated beef Campylobacter through the membrane on to the agar surface. A light
pieces (25 g) by ltration using mixed ester membranes after 24 h enrichment in centrifugation step was included to pull the heavier meat proteins and
Campylobacter enrichment broth (5% lysed horse blood), under an atmosphere of 2.5% particles to the bottom of the tube leaving the test bacteria in the
O2, 6% H2, 10% CO2 and balance N2. supernatant. The use of density gradient centrifugation on food
Species No. strains Mean counta samples has previously been demonstrated (Dos Santos Furtado and
included (Log10CFU g2) Casper, 2000; Fukushima et al., 2007). As the lter is the only selective
C. coli 3 8.06 defence against competing microora, the volume of supernatant
C. concisus 3 8.04 added was regulated to ensure that no spillage of the sample directly
C. curvus 3 8.18 onto the agar occurred during membrane removal. Examination of
C. fetus subsp. fetus 3 7.96
various supernatant aliquots ltered, showed that volumes greater
C. fetus subsp. venerealis 3 8.08
C. gracilis 2 8.05 than 300 l could not be fully adsorbed by the lter resulting in
C. helveticus 2 8.03 signicant run-off. It was concluded that 200 l of supernatant,
C. hominis 2 8.09 dropped at regular pattern intervals on the surface of the membrane
C. hyointestinalis subsp. hyointestinalis 2 8.1 enabled the greatest passage of Campylobacter while avoiding
C hyointestinalis subsp. lawsonii 2 8.16
membrane saturation and excess run-off.
C. jejuni subsp. doylei 3 8.21
C. jejuni subsp. jejuni 5 8.16 Contrary to reports that microora commonly associated with
C. lanienae 2 8.12 beef, such as E. coli and Proteus (Scanga et al., 2000; Reid et al., 2002;
C. lari 2 8.32 McEvoy et al., 2004; Kim et al., 2005) should not pass through the
C. mucosalis 2 8.05
lter due to their size and lack of motility at 37 C (Coetzee and de
C. rectus 2 7.9
C. showae 2 7.99 Klerk, 1968; Way et al., 2004; Bolster et al., 2006), some background
C. sputorum 3 8.28 interference was observed in this study. The most common contam-
C. upsaliensis 3 8.17 inant was identied as Lactobacillus using API 20E test strips
C. ureolyticus 2 8.17 (Biomrieux, France), which is easily distinguished from Campylo-
a
SED 0.023. bacter spp. based on colony morphology and gram reaction.
6 .A. Lynch et al. / Journal of Microbiological Methods 83 (2010) 17
Although some interspecies differences in colony size and colour
were noted in this study, colony morphology is not a reliable method
for differentiation between species. The pleomorphic nature of
Campylobacter means that slight difference in the moisture content
of agar (Buck and Kelly, 1981) or changes in growth atmosphere
(Jones et al., 1993) can result in major morphological difference
between colonies of the same strain.
This is the rst study to report a single cultural approach for both
the growth of a wide range of diverse Campylobacter species and their
recovery from articially inoculated meat. All isolates were conrmed
either by PCR (Bastyns et al., 1995; Klena et al., 2004; Personal
communication, Miller, W. G, Parker, C. T and Mandrell, R.E) or by use
of a biochemical test matrix (Le Roux and Lastovica, 1998). The
application of this method in a subsequent study will provide more
accurate data on the prevalence and diversity of Campylobacter
species in retail meats in the Republic of Ireland.
Acknowledgement
This work was funded under the E.U. Framework V: QLRT-CT-
2001-02201, (Campycheck).
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