ORIGINAL ARTICLE

Sources of the adventitious microflora of a smear-ripenedcheeseJ. Mounier1, S. Goerges2, R. Gelsomino3, M. Vancanneyt3, K. Vandemeulebroecke3, B. Hoste3,N.M. Brennan1, S. Scherer2, J. Swings3,4, G.F. Fitzgerald5 and T.M. Cogan1

1 Moorepark Food Research Centre, Teagasc, Fermoy, Ireland

2 Abteilung Mikrobiologie, Zentralinstitut fur Ernahrungs- und Lebensmittelforschung Weihenstephan, Technische Universitat Munchen, Freising,

Germany

3 BCCMTM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, Ghent, Belgium

4 Laboratorium voor Microbiologie, Ghent University, Ghent, Belgium

5 Department of Microbiology, University College, Cork, Ireland

Introduction

Surface-ripened cheeses are either mould-ripened, e.g.

Camembert and Brie, or bacterial-ripened, e.g. Reblochon,

Tilsit or Brick. The latter are also called smear-ripened

cheeses because of the development of a viscous, red-

orange smear of micro-organisms on their surfaces, which

are mainly responsible for the development of the flavour

characteristics of the cheese (Corsetti et al. 2001; Valdes-

Stauber et al. 1997) or washed-rind cheeses because they

are washed several times during ripening with dilute salt

solutions to spread the microflora. The ripening process

starts with the development of yeast, which metabolize

lactate to CO2 and H2O and form alkaline metabolites,

such as ammonia (Corsetti et al. 2001), which lead to the

deacidification of the cheese surface, enabling the growth

of salt-tolerant, but less acid-tolerant, Gram-positive,

catalase-positive bacteria, such as Staphylococcus sp. and

coryneforms.

Traditionally, the so-called ‘old young’ smearing tech-

nique, in which young cheeses are washed with a saline

suspension of micro-organisms from the surface of

Keywords

brine, Corynebacterium, Debaryomyces

hansenii, microflora, smear cheese,

Staphylococcus.

Correspondence

Timothy M. Cogan, Moorepark Food Products

Research Centre, Teagasc, Fermoy, Ireland.

E-mail: tcogan@moorepark.teagasc.ie

2005/0583: received 24 May 2005, revised 8

September 2005 and accepted 23 January

2006

doi:10.1111/j.1365-2672.2006.02922.x

Abstract

Aims: To determine the relationships between the major organisms from the

cheese-making personnel and environment and the surface of a smear cheese.

Methods and Results: 360 yeast and 593 bacteria from the cheese surface, the

dairy environment and the hands and arms of personnel were collected.

Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain

reaction and 16S rDNA sequencing were used for typing and identifying the

bacteria, and mitochondrial DNA restriction fragment length polymorphism

and Fourier-transform infrared spectroscopy for typing and identifying the

yeast. The three most dominant bacteria were Corynebacterium casei, Coryne-

bacterium variabile and Staphylococcus saprophyticus, which were divided into

three, five and seven clusters, respectively, by macrorestriction analysis. The

same clones from these organisms were isolated on the cheese surface, the dairy

environment and the skin of the cheese personnel. Debaryomyces hansenii was

the most dominant yeast.

Conclusions: A ‘house’ microflora exists in the cheese plant. Although the ori-

ginal source of the micro-organisms was not identified, the brines were an

important source of S. saprophyticus and D. hansenii and, additionally, the

arms and hands of the workers the sources of C. casei and C. variabile.

Significance and Impact of the Study: This is the first time that the major con-

tribution of the house microflora to the ripening of a smear-ripened cheese has

been demonstrated.

Journal of Applied Microbiology ISSN 1364-5072

668 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681

ª 2006 The Authors

mature cheeses, was used. Although it ensures the transfer

of desirable micro-organisms necessary for the ripening

process, it can also affect the transfer of spoilage or

pathogenic micro-organisms, such as Listeria monocyto-

genes (Valdes-Stauber et al. 1997). Commercial starter

cultures for cheese ripening are also often used to smear

the young cheeses. Such starter cultures, principally com-

posed of Brevibacterium linens, Debaryomyces hansenii and

Geotrichum candidum, may be sprayed on the cheese sur-

face, used as an inoculum to smear young cheeses or

added to the brine (Petersen et al. 2002). Recent studies

(Brennan et al. 2002; Petersen et al. 2002; Feurer et al.

2004) have shown that the yeast and/or bacterial ripening

starters do not necessarily develop on the cheese surface

and that instead, an adventitious flora, probably origin-

ating from the cheese-making environment, dominates

the cheese surface. Brennan et al. (2002), using a

polyphasic approach combining phenotypic, chemotaxo-

nomic and genotypic analyses, showed that Brevibacterium

aurantiacum BL2, despite being deliberately inoculated on

the cheese surface of Gubbeen cheese, was not a signifi-

cant member of the surface flora and no microbial suc-

cession of the bacterial species occurred during the

ripening. Indeed, single clones of novel species of Coryne-

bacterium casei, Corynebacterium mooreparkense and

Microbacterium gubbeenense dominated the cheese surface

microflora during the ripening period. Corynebacterium

mooreparkense has been shown to be a junior subjective

synonym for Corynebacterium variabile (Gelsomino et al.

2005). Feurer et al. (2004) showed that, B. linens, which

was heavily inoculated on the surface of a French smear-

ripened cheese in the initial stages of ripening, had

almost disappeared from the surface after 21 days and

that, after 31 days of ripening, a species of Arthrobacter

dominated the bacterial flora. More recently, other novel

species, Staphylococcus equorum ssp. linens, Staphylococcus

succinus ssp. casei (Place et al. 2002, 2003) and Arthro-

bacter arilaitensis and Arthrobacter bergerei (Irlinger et al.

2005) have been identified in smear-ripened cheeses.

Brevibacterium linens ATCC 9175, which was originally

isolated from Camembert cheese, has been shown to be a

member of a novel species B. aurantiacum (Gavrish et al.

2004) and Brevibacterium helvolum, a novel species of a

novel genus Pseudoclavibacter helvolus (Manaia et al.

2004).

There appears to be much less diversity in the dom-

inant yeast found on the surface of smear-ripened

cheeses. Debaryomyces hansenii and G. candidum have

been reported in rennet cheeses and Kluyveromyces

marxianus and Pichia membranifaciens in acid-curd

cheeses (Eliskases-Lechner and Ginzinger 1995; Petersen

et al. 2002; Valdes-Stauber et al. 1997). Petersen et al.

(2002) showed that a progression of D. hansenii strains

occurred during the ripening of a Danbo-type surface-

ripened cheese.

The studies cited above confirm the importance of an

adventitious, resident microflora in the ripening process

of red-smear cheeses and raise the question of the poss-

ible sources of those organisms in the cheese-making

environment and how they are transferred to the cheese

surface. To our knowledge, this aspect of cheese-ripening

has not been studied previously.

The aims of this study were to determine the relation-

ships between the major organisms on the skin of the

workers in the factory, in the cheese-making environment

and on the cheese surface using a polyphasic approach.

Material and methods

Cheese manufacture

Gubbeen cheese, a farmhouse, surface-ripened cheese sim-

ilar to Reblochon, Tilsit and Limburger cheeses, was stud-

ied. It is made from pasteurized milk with a mesophilic

starter culture. After brining, the surface of the cheese is

smeared with a saline suspension of a commercial ripen-

ing culture (OFR9, Visby, Neibull, Germany), which is a

mixture of yeast and bacteria, on three successive days,

and washed with a dilute salt solution over the next

3 days.

Microbiological examination

The surface microflora of each cheese was enumerated as

described previously by Brennan et al. (2002). Thirty bac-

terial and 50 yeast isolates were made at an early stage of

ripening (4 days), in the middle of ripening (10 days)

and at the late stage of ripening (16 days). Twenty bacter-

ial and 20 yeast were isolated from the commercial cul-

ture (OFR9, Visby) used in ripening.

Environmental samples were taken on day 3 of ripen-

ing, except the milk, which was sampled on the day of

manufacture (day 1) and the brine samples which were

taken on day 1. The cheese-making personnel were sam-

pled during their working duties. Samples from each of

the three brines, milk in the vat after pasteurization, tap

water, water (previously boiled) used to prepare the

smearing suspension and the saline suspension contain-

ing the ripening starters were also examined. All surfaces

were sampled using a wet swabbing technique. A sterile

cotton swab (Copan, Brescia, Italy) was moistened by

immersion in 10 ml of sterile Maximum Recovery Dilu-

ent (Oxoid) in a 20 ml sterile container. Excess moisture

was removed by compression of the swab against the

inner wall of the container and the swab was rubbed

across the sampling site. The swab tip was then broken

J. Mounier et al. Sources of the adventitious microflora

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681 669

off into the sterile container containing the wash fluid.

The wired stainless steel shelves and the lower arms,

hands and/or gloves of the workers were sampled with-

out any delimited surface area to ensure maximal recov-

ery. An area of 10 · 10 cm of a wooden shelf was also

examined. The microflora of each sample was enumer-

ated on Milk Plate Count Agar containing 5% salt (bac-

teria) and Glucose Chloramphenicol Agar (yeast) and

isolates were taken from the highest dilution. The air in

the manufacturing and curing room was sampled using

an air sampler (Anderson, Smyrna, GA, USA) with an

exposure time of 3 min on the two media cited above.

All the isolates were purified by restreaking twice and

were stored at )80�C in a 1 : 1 mixture of Trypticase Soy

Broth (TSB) and glycerol.

Phenotypic characterization of bacteria

Bacterial cultures were Gram-stained and tested for the

presence of catalase. The cell morphology of each isolate

was determined under phase microscopy on mineral base E

Yeast Extract Glucose Agar (EYGA) after incubation at

30�C for 8 h, 1, 3 and 7 days as described by Cure and

Keddie (1973). Strains that were Gram-positive, catalase-

positive, irregularly rod-shaped or that underwent a

rod/coccus transformation were considered to be coryne-

forms and were submitted to pulsed-field gel electro-

phoresis (PFGE) analysis as described below. The

Gram-positive, catalase-positive cocci strains were tested

for their ability to grow anaerobically in a glucose-contain-

ing medium, to grow aerobically in the presence of glycerol

(10 g l)1) and erythromycin (0Æ4 mg l)1) and tested for

their susceptibilities to lysozyme (400 lg ml)1), lysosta-

phin (200 lg ml)1) and furazolidone (100 lg diffusion

disc; Evans and Kloos 1972; Kloos and Schleifer 1975) and

were submitted to PFGE analysis as described below.

PFGE analysis

Pulsed-field gel electrophoresis was performed using a

method described previously by Brennan et al. (2002).

Lysis of all coryneform isolates except coccal isolates con-

sisted of one step in 10 mg ml)1 lysozyme. Staphylococcus

sp. were lysed first overnight at 37�C in a mixture of

10 mg ml)1 lysozyme and 50 lg ml)1 lysostaphin fol-

lowed by a further overnight incubation at 37�C in

10 mg ml)1 lysozyme. The restriction enzymes used were

SmaI for the Staphylococcus sp. and SpeI for the

coryneform isolates. Digested chromosomal DNA of

coryneform strains presenting patterns with mainly low-

molecular weight fragments was digested using AscI. Gels

were stained using ethidium bromide (0Æ5 lg ml)1) for

2 h, destained in water and photographed using a digital

camera. The digitized patterns were normalized and ana-

lysed numerically using bionumerics, version 2.0,

(Applied Maths, Sint-Martems-Latem, Belgium). Similar-

ities among band patterns were calculated based on Pear-

son’s similarity coefficient and dendrograms were built

using UPGMA (unweighted pair group method with

arithmetic mean).

Repetitive sequence-based PCR genomic fingerprinting

One to three representatives of the strains of each PFGE

cluster and reference strains were submitted to repetitive

sequence-based polymerase chain reaction (rep-PCR) ge-

nomic fingerprinting. Biomass was scraped from Trypti-

case Soy Agar incubated for 1 day, suspended in 1 ml of

TE 10/1 [10 mmol l)1 Tris-HCl, 1 mmol l)1 ethylenedi-

aminetetraacetic acid (EDTA), pH 8Æ0] and centrifuged

for 10 min at 2700 g at 4�C. Cells were then kept for at

least 1 h at )20�C. Total DNA was extracted using a

modification of the method described by Gevers et al.

(2001). The lysis buffer contained 1330 U ml)1 mutanoly-

sin and 40 mg ml)1 lysozyme for rod-shaped isolates and

1330 U ml)1 lysostaphin and 40 mg ml)1 lysozyme for

coccal isolates. The oligonucleotide primers used were

BOXA1R (5¢-TACGGCAAGGCGACGCTACG-3¢) for

the Gram-positive, catalase-positive rods and (GTG)5

(5¢-GTGGTGGTGGTGGTG-3¢) for the Gram-positive,

catalase-positive cocci, each with the appropriate PCR

programme (Versalovic et al. 1994). PCR amplifications

were performed with a DNA thermal cycler (Perkin-El-

mer, model 9600; Milan, Italy) as described previously

(Versalovic et al. 1994), using Goldstar DNA polymerase

(Eurogentec, Seraing, Belgium). The PCR products were

run in a 1Æ5% (w/v) agarose gel for 16 h at a constant

voltage of 55 V in 1X TAE (40 mmol l)1 Tris-acetate,

1 mmol l)1 EDTA, pH 8Æ0) at 4�C. The rep-PCR patterns

were visualized after staining in ethidium bromide solu-

tion (1 lg ml)1) under UV light and digitized using a

digital camera. The resulting fingerprints were analysed

using bionumerics. Similarities among band patterns

were calculated based on Pearson’s similarity coefficient

and dendrograms were built using UPGMA.

16S rDNA sequencing

Partial sequencing (approximately 700 bp) of the 16S

rDNA gene was performed on the strains that remained

unidentified after rep-PCR fingerprinting as described

by Vancanneyt et al. (2005). Sample preparation was

assisted using a Tecan Genesis Workstation 200 (Tecan,

Mannedorf, Switzerland). Sequence assembly was per-

formed using AutoAssembler (Perkin-Elmer). The closest

related sequences were found using the fasta program.

Sources of the adventitious microflora J. Mounier et al.

670 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681

ª 2006 The Authors

Fourier-transform infrared spectroscopy

Fourier-transform infrared (FTIR) spectroscopy was per-

formed to identify yeast isolates as described previously

by Kummerle et al. (1998). For recording and evaluating

the spectra, an IFS-28B FTIR spectrometer and the opus

software for Windows, version 3.17 (Bruker, Ettlingen,

Germany), were used. The used yeast database comprises

around 2500 reference spectra.

Mitochondrial DNA restriction fragment length poly-

morphism of D. hansenii strains

Total DNA was extracted according to a modification of

the methods described previously by Romano et al.

(1996) and Petersen et al. (2002). Yeast cells, grown in

5 ml of YEPD (1% yeast extract, 2% peptone, 2%

glucose, pH 6Æ0) at 25�C overnight, were harvested,

washed with 1 ml of distilled water, harvested again and

resuspended in 0Æ5 ml of solution A (0Æ9 mol l)1 sorbitol,

0Æ1 mol l)1 EDTA, pH 7Æ5, 200 lg ml)1 lyticase). The

mixture was incubated for 60 min at 37�C to produce

spheroplasts. These were harvested at 5200 g for 5 min,

resuspended in 0Æ5 ml of solution B [50 mmol l)1 Tris-

HCl, 20 mmol l)1 EDTA, 1% w/v sodium dodecyl sulfate

(SDS), pH 7Æ5, 100 lg ml)1 proteinase K] and incubated

30 min at 65�C. Then, 200 ll of 5 mol l)1 sodium acetate

were added and the mixture placed on ice for 30 min.

After centrifugation at 10 600 g for 15 min, the superna-

tant was precipitated with one volume of isopropanol for

15 min at room temperature and centrifuged for 10 min

at 20 800 g. The supernatant was decanted and the DNA

washed with 70% ethanol and centrifuged for 5 min at

20 800 g. The residual ethanol was aspirated with a pip-

ette and the pellets were dried on a heating block at 37�C

for 45 min. The dried DNA was dissolved in 25 ll TE 10/

1. About 10–15 ll of the purified DNA was digested with

5 U of HaeIII overnight at 37�C. The restriction frag-

ments were analysed by electrophoresis on a 1% (w/v)

agarose gel in 1X TAE buffer at 100 V for 3 h. kDNA cut

with HindIII was used as a marker. The restriction frag-

ments were visualized by ethidium bromide staining and

UV transillumination. After photography using a digital

camera, the resulting fingerprints were analySed using

bionumerics software. Similarities among band patterns

were calculated based on Dice’s similarity coefficient and

a dendrogram was built using UPGMA.

Bacterial strains deposit

Representatives of the major PFGE clones of Corynebacte-

rium casei, Corynebacterium variabile and Staphylococcus

saprophyticus, isolated in this study, were deposited in the

BCCM/TM/LMG bacteria collection (Laboratory of

Microbiology, Ghent University, Belgium). Their respect-

ive LMG numbers are C. casei LMG 23048, 23047, 23046

(clusters 4–6, Fig. 4), C. variabile LMG 23052, 23051,

23053, 23050, 23049 (clusters 11–15, Fig. 5) and

S. saprophyticus LMG 23039, 23040, 23041, 23042, 23043,

23044, 23045 (clusters 5–11, Fig. 6).

Chemical analysis of cheese surface

Moisture and salt were determined by standard methods

(Lynch et al. 1997). pH was determined by placing the

electrodes directly into the grated cheese.

Results

Composition of the cheese surface

The surface pH increased from 4Æ9 to 6 between days 4

and 16 while the moisture content decreased from 44Æ7%

to 40Æ2% between days 4 and 10 after which it remained

constant to day 16 (data not shown). The salt content

ranged between 1Æ9% and 2Æ2% (w/v). The increase in the

salt-in-moisture content between days 10 and 16 coin-

cided with a loss of moisture from the cheese surface

(data not shown). The salt content in the brine was 23%

(w/v, data not shown).

Composition of OFR9 culture

Two different morphotypes of bacteria were found, one

producing an orange pigment and the other a white

one. Ten isolates of each morphotype were analysed by

PFGE and each gave identical patterns, implying that

only two bacterial species were present. The white iso-

lates were identified as P. helvolus, and the orange iso-

lates as Brevibacterium spp. Twenty yeast isolates all

gave the same mitochondrial DNA (mtDNA) restriction

fragment length polymorphism (RFLP) pattern and

could not be identified from the ca. 2500 yeasts in the

FTIR database.

Microbiology of cheese surface and environment

The bacterial and yeast counts on the cheese surface dur-

ing ripening, in the milk and in the environment, are

illustrated in Fig. 1a,b respectively.

The bacterial counts on the cheese surface increased

from 4 · 104 to 2 · 107 CFU cm)2 between days 4 and

10 of ripening and remained constant between days 10

and 16. The yeast counts increased only slightly from

105 CFU cm)2 on day 4 to 106 CFU cm)2 on day 16

(Fig. 1a).

J. Mounier et al. Sources of the adventitious microflora

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681 671

Yeast counts were approximately 104 CFU ml)1 in the

diluted smear suspension used to smear the young

cheeses and approximately 105 CFU ml)1 in the brines

(Fig. 1b). Counts of approximately 104 CFU cm)2 were

found on the wooden shelf. The count of yeast in the

swab of the stainless steel shelves was 6Æ9 · 103 CFU ml)1

(data not shown). Yeast (<10 CFU ml)1) were not detec-

ted in the pasteurized milk in the cheese vat, in the tap

water or in the water used to prepare the diluted smear

suspension. Five yeast were found in the air of the curing

room and none in the air of the manufacturing room

(data not shown).

Bacterial counts in the dairy environment are illustra-

ted in Fig. 1b. Counts of approximately 102 CFU ml)1

were found in the tap water, water used to prepare the

diluted smear suspension and in the pasteurized milk.

The bacterial counts in the brines and in the diluted

smear suspension were approximately 104 CFU ml)1 and

106 CFU ml)1 respectively. The surface of the wooden

shelves had counts of approximately 105 CFU cm)2. The

bacterial count in the swab of the stainless steel shelves

was 105 CFU ml)1 (data not shown). Two hundred and

ninety-four and 150 bacteria were found in the air of the

manufacturing and curing room, after 3 min of exposure

respectively (data not shown).

Counts in the swabs of the skin or gloves surface ran-

ged from 4Æ9 · 102 to 2Æ4 · 106 CFU ml)1 for the bac-

teria and from <10 CFU ml)1 to 1Æ5 · 106 CFU ml)1 for

the yeast (Table 1). The bacterial counts of the hand and

arm swabs of each worker varied between 4Æ9 · 102 and

2Æ4 · 106 CFU ml)1 whereas the yeast counts varied

between <10 and 1Æ7 · 105 CFU ml)1 in the hand and

arm swabs. Although the areas of the hands and arms,

which were swabbed, were not delimited, the counts are

reported per millilitre of diluent to allow comparisons to

be made.

Bacterial identification

All salt-tolerant bacteria were Gram-positive, catalase-

positive micro-organisms. All were examined by PFGE and

one to three representatives of each cluster were submitted

to rep-PCR analysis. From an overall total of 593 bacteria,

545 were identified using this technique, 224 coryneform

bacteria using the BOX-primer, and 301 staphylococci

using the (GTG)5-primer (data not shown). Nine species,

C. casei, C. variabile, Agrococcus sp., M. gubbeenense,

P. helvolus, B. linens, A. arilaitensis, S. saprophyticus and

S. equorum, were identified. Twenty-six isolates were iden-

tified using 16S rDNA partial sequencing because the

rep-PCR failed to give a categorical identification. Twenty-

one isolates were not identified.

A progression of species was found during ripening

(Fig. 2). Staphylococcus saprophyticus dominated the

cheese surface microflora at the early stage of ripening

with 29 of 30 isolates. The other isolate was identified as

P. helvolus, one of the micro-organisms deliberately

inoculated on the cheese surface. However, this species

was not recovered at the middle and the late stages of

ripening. Instead, C. variabile was the dominant species,

followed by C. casei. The former increased from four to

10 of 30 isolates and the latter decreased from 21 to 15 of

30 isolates, respectively, from mid- to late-ripening. Only

two and one of 30 isolates of S. saprophyticus were found

at the mid- and late-stages. Agrococcus sp. was also isola-

ted from the cheese surface at the middle and the late

Early (4 days) Middle (10 days) Late (16 days)

Stage of ripening (days)

CF

U c

m–2

104

107

106

105

104

103

102

101

106

107

108(a)

(b)

105

Milk

Tap

water

Smea

r wat

er

Smea

r sus

pens

ion

Brine

1

Brine

2

Brine

3

Woo

den

shelf

CF

U m

l–1 o

r C

FU

cm

–2

Figure 1 Bacterial (grey bars) and yeast (black bars) counts in the

surface layer of the cheese during ripening (a) and in the dairy envi-

ronment at 3 days of ripening (b).

Table 1 Bacterial and yeast counts of the hands, arms and gloves of

the workers

Worker

Hands (CFU ml)1) Arms (CFU ml)1) Gloves (CFU ml)1)

Bacteria Yeast Bacteria Yeast Bacteria Yeast

W1 4Æ9 · 102 <10 4Æ4 · 104 1Æ7 · 105 3Æ5 · 104 1Æ5 · 106

W2 2Æ4 · 106 3Æ3 · 104 1Æ8 · 106 1Æ6 · 105

W3 7Æ8 · 104 <10

W4 4Æ2 · 104 2Æ2 · 103

W5 7Æ5 · 103 2Æ2 · 101 4Æ4 · 103 3Æ3 · 102

W6 1Æ8 · 105

Sources of the adventitious microflora J. Mounier et al.

672 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681

ª 2006 The Authors

stage of ripening with two and one of 30 isolates respect-

ively. Two isolates were not identified.

A larger bacterial diversity was found on the skin

surface and gloves of the workers. Of 266 isolates, 259

isolates were Gram-positive, catalase-positive organisms

and one was a Gram-negative, catalase-positive rod, and

18 different bacterial species or genera were identified

(Table 2). Three to six different species were identified

in each sample except for the hands of worker five from

which 13 different bacteria species were isolated. Staphy-

lococcus saprophyticus was the dominant micro-organism

isolated from the skin surface representing 42% of the

isolates and was recovered from eight of nine workers,

followed by C. casei (24Æ4%, eight samples), C. variabile

(7Æ5%, seven samples), M. gubbeenense (2Æ6%, four

samples) and Agrococcus sp. (2Æ8%, two samples).

Pseudoclavibacter helvolus (7Æ5%, two samples), a strain

used as a ripening starter, was recovered as well but

those strains were only found on the gloves and on the

arms of the worker who was smearing the young cheeses

with the commercial ripening starter. The other organ-

isms were isolated sporadically. Six isolates were not

identified.

In the dairy environment, 156 isolates (84%) were

identified as S. saprophyticus (Table 3). It was the only

micro-organism in the air, the water used to prepare the

smear suspension, the milk from the vat, the tap water

and was the dominant micro-organism in the brining

baths. Very few organisms were identified on the stainless

steel shelves while C. casei, C. variabile, M. gubbeenense

and Agrococcus sp. were found on the surface of the woo-

den shelf. Pseudoclavibacter helvolus was only isolated

from the diluted smear suspension. Thirteen isolates, all

from the brines, were not identified.

0

5

10

15

20

25

30

S. sap

roph

yticu

s

C. cas

ei

C. var

iabile

Agroc

occu

s sp.

P. he

lvolus

Uniden

tified

Num

ber

of is

olat

es

Figure 2 Progression of the bacterial species on the cheese surface

after 4 (black bars), 10 (grey bars) and 16 (white bars) days of ripen-

ing.

Table 2 Distribution of the bacterial species isolated from the hands, arms and gloves of the workers

Bacterial species

Arms Hands Gloves

TotalW1* W2 W1 W2 W3 W4 W5 W6 W1

Corynebacterium casei 3 4 3 15 13 2 5 20 65

C. variabile 4 2 3 2 1 4 2 18

C. testudinor� (AJ969167) 1 1

Microbacterium gubbeenense 2 3 1 1 7

M. oxydans� (AJ969168) 2 2

Arthrobacter arilaitensis 1 1

A. mysorens� (AJ967024) 5 5

Brevibacterium linens 1 2 3

Pseudoclavibacter helvolus 1 19 20

Agrococcus sp. 11 2 13

Micrococcus luteus� (AJ969169) 1 1

Kocuria sp.� (AJ969170) 5 5

Staphylococcus saprophyticus 22 23 24 3 26 1 2 10 112

S. pasteuri� (AJ969171) 1 1

S. equorum 2 1

S. epidermidis� (AJ969172) 1 1

Rothia sp.� (AJ969174) 3 3

Psychrobacter sp.� (AJ969173) 1 1

Unidentified 1 2 1 1 1 6

Total 30 30 29 30 30 30 27 30 30 266

*Each number corresponds to a different worker.

�Marked strains were additionally analysed by partial sequence of the 16S rDNA (presumed identification) and their respective 16S rDNA partial

sequence accession numbers in EMBL is indicated in parentheses.

J. Mounier et al. Sources of the adventitious microflora

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681 673

Intraspecies bacterial diversity

The intraspecies diversity among the coryneforms isolated

from the cheese surface and in the cheese-making environ-

ment, examined using PFGE, and the patterns of the two

bacterial ripening starters are illustrated in Figs 3 and 4.

Two restriction enzymes, AscI and SpeI, were used to

improve strain differentiation. As can be seen in Fig. 3,

three different clones of C. casei (clusters 4–6) were isolated

and two of those clones (clusters 4 and 5) were found on

the cheese surface, the skin of the workers and the wooden

shelves. One clone (cluster 6) was only isolated from the

skin surface. Five clones of C. variabile were isolated

(Fig. 4). One clone of C. variabile (cluster 11) was only

found on the cheese surface and two clones (clusters 13

and 15) were found on the cheese surface, the workers’ skin

and the wooden shelves. One clone (cluster 14) was only

isolated from the skin surface. The other micro-organisms

identified showed only one clone except B. linens for which

two clones were found (clusters 8 and 10).

The intraspecies diversity among the staphylococci

using PFGE is illustrated in Fig. 5. Six clones of

S. saprophyticus were found (clusters 5–11). Three clones,

clusters 5–7, were only found on the skin surface of the

workers while the others were isolated from the cheese

surface during ripening, the skin and gloves of the work-

ers and the dairy environment (brine, milk, stainless steel

shelves and air). These clones had highly similar SmaI

restriction patterns that differed in only one or two

bands. One clone of Staphylococcus equorum (cluster 1),

two clones of Staphylococcus sp. showing 100% similarity

with S. epidermidis (clusters 3 and 4) and one clone of

Staphylococcus sp. showing 100% similarity with S. paste-

uri (cluster 2) on the basis of 16S rDNA partial sequen-

cing, were found.

Identification of the yeast species

The results of the identification and the distribution of

the different yeast species, isolated from the cheese sur-

face, the skin and the gloves of the workers and in the

dairy environment, are shown in Table 3. Debaryomyces

hansenii was found to dominate the cheese surface during

ripening and was the most abundant species, with 328

(91%) of 360 isolates, isolated from the workers and in

the dairy environment (brine, air, stainless steel shelves).

Other species were found only occasionally, Candida ca-

tenulata and Candida intermedia on the cheese surface

and Clavispora lusitaniae, Rhodotorula glutinis and Yarr-

owia lipolytica from the skin surface. The 20 isolates of

the OFR9 culture, which were analysed, clustered together

but did not cluster with any other yeast isolated in this

study (data not shown). This species could not be identi-

fied using the database of the FTIR reference spectra.

Strain typing of D. hansenii

Three hundred and twenty-eight D. hansenii strains were

typed using mtDNA RFLP (Fig. 6). The intraspecies

diversity was very low with one highly dominant RFLP

Table 3 Distribution of the bacterial species isolated from the dairy environment

Bacterial species

Air samples Liquid samples

Ripening

shelves

TotalMR CR I W TW M B1 B2 B3 WS SS

Corynebacterium casei 11 11

C. variabile 6 6

Microbacterium gubbeenense 3 3

Brevibacterium linens 1 2 3

Pseudoclavibacter helvolus 28 28

Curtobacterium sp.* (AJ967025) 1 1 2

Brachybacterium sp.* (AJ969175) 1 1

Agrococcus sp. 10 10

Staphylococcus saprophyticus 30 30 2 12 6 6 26 19 25 1 157

S. epidermidis* (AJ969172) 2 2

Dietzia sp.* (AJ969176) 1 1

Unidentified 1 8 4 13

Total 30 30 30 12 6 6 30 30 30 30 3 237

MR, air of the manufacturing room; CR, air of the curing room; I, diluted inoculum; W, water used to dilute inoculum; TW, tap water; M, milk

out of vat; B1, 2 and 3, brine baths 1, 2 and 3; WS, wooden shelves; SS, stainless steel shelves.

*Marked strains were additionally analysed by partial sequence of the 16S rDNA (presumed identification) and their respective 16S rDNA partial

sequence accession numbers in EMBL is indicated in parentheses.

Sources of the adventitious microflora J. Mounier et al.

674 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681

ª 2006 The Authors

profile (323 out of 328). The same RFLP profile was

found for the D. hansenii strains isolated from the cheese

surface throughout the ripening period, which means that

no succession of D. hansenii strains occurred during

ripening. The strain, which colonized the cheese surface,

had the same RFLP profile as the one found in the brine,

on the stainless steel shelves and on the hands/arms and

gloves of the workers. The absence of implantation of the

yeast from the commercial starter was confirmed using

mtDNA RFLP. Indeed, except in the diluted smear sus-

pension, none of the yeast isolated in this study had a

similar RFLP profile (Fig. 6).

Discussion

In this study, 360 yeast and 593 bacteria isolated from the

surface of an Irish smear-ripened cheese during ripening,

from the dairy environment and from the hands and

arms of the cheese personnel were identified using pheno-

typic and genotypic techniques; one yeast and 21 (3Æ5%)

bacterial isolates were not identified. Twenty bacterial

(Tables 2 and 3, and Fig. 2) and six yeast species were

identified (Table 4). To our knowledge, this is the first

time a comparative study of the bacterial and yeast flora

on the cheese surface during ripening and in the dairy

environment has been undertaken.

The three most dominant bacteria were C. casei,

C. variabile and S. saprophyticus and these were divided

into three, five and seven clusters, respectively, by macro-

restriction analysis. In addition, a single clone of Agrococ-

cus spp. was also present on the hands of some workers

and in the cheese at the mid- and late-stages of ripening.

It is generally believed in epidemiological studies that

strains of a particular species showing up to three differ-

ent bands are in fact the same clone (Tenover et al.

1997). On this basis, a single clone of C. casei (Fig. 3)

and probably two each of C. variabile and S. saprophyticus

were present, with one clone of C. variabile (clusters 13–

15 inclusive, Fig. 4) and S. saprophyticus (clusters 7–11

inclusive, Fig. 5) being dominant. These dominant clones

contained isolates from most sources. The RFLP profiles

of the mtDNA of 323 isolates of D. hansenii were identi-

cal implying but not proving that they are also closely

related if not identical clones.

The same clones of S. saprophyticus were found at the

early stage of cheese ripening, in the milk, although only

six isolates were made from it, the environment including

the tap water, the brine, the air in the curing room and

100 95 90 85 80 75 70 65 60 55

Similarity (%)

Cluster 1: W2*H (5)

Cluster 2: W5H (1)

Cluster 3: W5H (3)

A. mysorens †

Cluster 4: W1H (2), W2H (5), W3H (8), W4H (1), W5H (2), W6H (12), W2A (2), WS (7), CL (7)

Cluster 5: W1H (1), W2H (10), W3H (5), W4H (1), W6H (7), W1A (3), W2A (2), WS (4), CM (4), CL (3)

C. casei

Cluster 6: W5H (3), W6H (1)

A. arilaitensis

Rothia sp. †

Number of isolates

5

1

3

6 4

40

4

Taxon

Figure 3 AscI restriction patterns of the coryneforms and results of their repetitive sequence-based polymerase chain reaction (rep-PCR) identifi-

cation using BOX-primer. A., Arthrobacter; C., Corynebacterium; CE, cheese at the early stage of ripening (4 days); CM, cheese at the middle

stage of ripening (10 days); CL, cheese at the late stage of ripening (16 days); WA, arms; WH, hands; WG, gloves; MR, air of the manufacturing

room; CR, air of the curing room; I, smear suspension; B1, 2 and 3, brine baths 1, 2 and 3; WS, wooden shelves; SS, stainless steel shelves. *Each

number corresponds to a different worker. �Marked strains were additionally analysed by partial sequence of the 16S rDNA (presumed identifica-

tion). Numbers in parentheses indicate the number of strains of that PFGE profile isolated.

J. Mounier et al. Sources of the adventitious microflora

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681 675

the arms and hands of the personnel. The strain of

D. hansenii isolated from the cheese surface was also

found in the brines, the air in the curing room and on

the hands and arms of the workers. Furthermore, the

same clones of C. casei and C. variabile were found on

the cheese, the wooden shelf and on the hands and arms

of the workers but not the milk, the brine or other envi-

ronmental samples. Similarly the same clone of Agrococcus

sp. was present on the hands of two workers and the

cheese at mid- and late-stages of ripening (Fig. 4). The

cheese receives considerable manual handling during the

early stages of ripening as each individual cheese is

washed and inoculated several times with a commercial

ripening culture. Except for one isolate of P. helvolus, the

components of the commercial culture were not recov-

ered from any cheese. All of this data suggest that a

‘house’ microflora is present not only in the ripening

rooms but also throughout the cheese-making premises,

which is transferred to the cheese surface from the brines

and/or from the hands of the personnel during cheese-

making and ripening. In this sense, the mode of inocula-

tion is like the ‘old-young’ inoculation, which is used in

smear cheese production in some countries. The original

source of this microflora was not identified but cheese-

specific clones of the same species have been found in

other washed-rind cheeses (Mounier et al. 2005). It is

therefore highly probable that the particular environmen-

tal and nutritional conditions on the cheeses surface select

for these particular species.

The milk for cheese-making was pasteurized, which

should kill any staphylococci present in it, yet six isolates

of the same clone S. saprophyticus were isolated from it

Cluster 1: W3*H (11), W6H (2),WS (10), CM (2), CL (1)

Cluster 2: Ripening starter “OFR9.2”

Cluster 3: W5H (1)

Agrococcus sp. †

Brevibacterium sp. †

M. oxydans †

Cluster 4: W5H (1) M. oxydans †

Cluster 5: B2 (1) Dietzia sp. †

Cluster 6: W2H (2), W3H (3),W5H (1), W6H (1), WS (3)

M. gubbeenense

Cluster 7: W1G (19), W1A (1),I (28), CE (1), ripening starter “OFR9.1”

P. helvolus

Cluster 8: W5H (1), W6H (2), B1 (1),B2 (1)Cluster 9: B1 (1), B3 (1)

Cluster 10: B2 (1)

B. linens

Cluster 11: CM (5), CL (6)

Cluster 12: W2H (1), W4H (1) ,W5H (1), W1A (2), CL (2)

Cluster 13: W2H (2), W6H (2),W2A (1), CM (8)

Cluster 14: W5H (3)

Cluster 15: W3H (2), W1A (2),W2A(1), WS (6), CM (9), CL (8)

C. variabile

30 40 50 60 70 80 90 100Similarity (%)

B. linens

Curtobacterium sp. †

Numberof isolates

26

1

1

1

10

49

Taxon

5

2

1

7

11

13

3

28

Figure 4 SpeI restriction patterns of the coryneforms and results of their repetitive sequence-based polymerase chain reaction (rep-PCR) identifi-

cation using BOX-primer. M., Microbacterium; P., Pseudoclavibacter; C., Corynebacterium; B., Brevibacterium; CE, cheese at the early stage of

ripening (4 days); CM, cheese at the middle stage of ripening (10 days); CL, cheese at the late stage of ripening (16 days); WA, arms; WH, hands;

WG, gloves; MR, air of the manufacturing room; CR, air of the curing room; I, smear suspension; B1, 2 and 3, brine baths 1, 2 and 3; WS, woo-

den shelves; SS, stainless shelves. *Each number corresponds to a different worker. �Marked strains were additionally analysed by partial sequence

of the 16S rDNA (presumed identification). Numbers in parentheses indicate the number of strains of that PFGE profile isolated.

Sources of the adventitious microflora J. Mounier et al.

676 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681

ª 2006 The Authors

Similarity (%)

Cluster 1: W1*H (2)

Cluster 2: W5H (1)

Cluster 3: SS (1)

Cluster 4: SS (1), W6H (1)

Cluster 6: W1H (2), W4H (4)

Cluster 7: W4H (16)

Cluster 8 : W1A (1), MR (30), CR(30), W (12), TW (6), I (2), CE (1)

Cluster 9: W1G (10), W4H (2), B1(25), CM (2), CL (1)

Cluster 10: W2A (21), W2H (3),W5H (1), W6H (2), M (6), SS (1),B1 (1)

Cluster 11: W1A (20), W1H (22),W4H (4), B2 (19), B3 (25), CE (28)

Cluster 5: W1A (1), W2A (2)

S. equorum

S. pasteuri †

S. epidermidis †

S. epidermidis †

S. saprophyticus

100908070605040

Numberof isolates

Taxon

2

1

1

3

2

6

16

82

40

35

118

Figure 5 SmaI restriction patterns of the staphylococci and results of their repetitive sequence-based polymerase chain reaction (rep-PCR) identifi-

cation using BOX-primer. S., Staphylococcus; CE, cheese at the early stage of ripening (4 days); CM, cheese at the middle stage of ripening

(10 days); CL, cheese at the late stage of ripening (16 days); WA, arms; WH, hands; WG, gloves; MR, air of the manufacturing room; CR, air of

the curing room; I, smear suspension; W, water to dilute commercial starter; TW, tap water; B1, 2 and 3, brine baths 1, 2 and 3; WS, wooden

shelves; SS, stainless steel shelves. *Each number corresponds to a different worker. �Marked strains were additionally analysed by partial

sequence of the 16S rDNA (presumed identification). Numbers in parentheses indicate the number of strains of that PFGE profile isolated.

Similarity (%) Strain designation

SS (1)

CR (2), W5*A (2)

OFR9 yeast starter

CE (47), CM (48), CL (45), W1G (12), W2A (10), W5A (2), W2H (19), W4H (14), W5H (3), CR (2), B1 (30), B2 (30), B3 (30), WS (2), SS (29)

100 50

Figure 6 Mitochondrial DNA restriction fragment length polymorphism (RFLP) profiles of Debaryomyces hansenii isolates from Gubbeen cheese

during ripening, the cheese-making environment and OFR9 yeast starter culture. CE, cheese at the early stage of ripening (4 days); CM, cheese at

the middle stage of ripening (10 days); CL, cheese at the late stage of ripening (16 days); WA, arms; WH, hands; WG, gloves; CR, air of the cur-

ing room; I, smear suspension; B1, 2 and 3, brine baths 1, 2 and 3; WS, wooden shelves; SS, stainless steel shelves. *Each number corresponds

to a different worker. Numbers in parentheses indicate the number of strains of that PFGE profile isolated.

J. Mounier et al. Sources of the adventitious microflora

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681 677

and from the tap water. This finding reinforces the idea

that a ‘house’ microflora is present.

Staphylococcus saprophyticus is a highly salt-tolerant

micro-organism (Kunin and Rudy 1991) and is therefore

able to survive in the brine. However, the dominant

strain isolated in the present study is unable to grow on

the surface of experimental cheeses produced under

aseptic conditions in the absence of D. hansenii probably

because of the low pH (5Æ2) on the surface of the cheese

after cheese manufacture (unpublished data). This result

also implies that its dominance on the cheese surface at

the early stage of ripening is mainly due to the high

numbers present in the brine and on the skin of the

personnel. Staphylococcus saprophyticus is a class 2 organ-

ism causing urinary tract infections in women (Hedman

et al. 1990). This raises the question of the safety status

of the cheese. This cheese has not been incriminated as

the cause of human disease. Staphylococcus saprophyticus

has also been isolated from other cheeses (Vernozy-Roz-

and et al. 1996; Irlinger et al. 1997; Delbes and Montel

2005) and from the surface of fermented meat products,

where the curing process and the salt are considered to

be the main sources of contamination (Vilar et al. 2000;

Gardini et al. 2003). Thus, the organism would appear

to be common in fermented foods. Eighty-three strains

from human skin were characterized by Schleifer and

Kloos (1975) and the organism is also considered to be

a transient species on human skin (Kloos and Mussel-

white 1975). In the cheese production unit, the skin of

the workers is permanently exposed to the organism

because of its extensive presence in the dairy environ-

ment. This results in permanent carriage of this species

and allows potential contamination from the skin to the

cheese surface. It would be interesting to study the

pathogenic properties of isolates from cheese, meat and

urinary tract infections.

The personnel were not swabbed prior to the start of

work so that it is not possible to say whether the organ-

isms isolated colonized the arms and hands of the person-

nel. Except for worker 5, who is only involved in cheese

manufacture, all personnel interchange their duties during

demoulding, brining and washing the cheeses during

ripening. This explains the presence of the same clones of

C. variabile, C. casei, S. saprophyticus and D. hansenii on

the arms and hands of the personnel and the cheese sur-

face.

The wooden shelves were also found to be a niche

not only for these particular micro-organisms but also

M. gubbeenense, which has been isolated previously from

Gubbeen cheese (Brennan et al. 2002). The cheeses were

not placed on the wooden shelves until day 18, which

was several days after the last cheese was sampled. Thus,

the wooden shelves per se were not the source of the

organisms in the present study. However, prior to the

introduction of the stainless steel shelves, the wooden

shelves were used from the beginning of ripening and

could act as a source. The use of stainless steel shelves

instead of the wooden shelves, despite the improved

hygienic standards, removes a potential source of the

organisms that develop on the cheese surface; neverthe-

less, one isolate of S. saprophyticus and 30 isolates of

D. hansenii were obtained from the stainless steel shelves.

Petersen et al. (2002) studied the yeast component of a

Danbo-type smear-ripened cheese during ripening and

showed that only one strain of D. hansenii was present

from day 3 of ripening. This was confirmed in the present

study where the same strain of D. hansenii dominated on

the cheese surface throughout the ripening period and in

the dairy environment. This organism is typically found

in cheese brines (Seiler and Busse 1990). The present

results are also consistent with those of Welthagen and

Viljoen (1998), who studied the yeast profile during pro-

Table 4 Distribution of the yeast species isolated on the cheese surface, the skin and gloves of the workers and in the dairy environment

Source CE CM CL W1G* W2A W5A W2H W4H W5H CR B1 B2 B3 I WS SS Total

Debaryomyces hansenii 47 48 45 12 10 4 19 14 3 4 30 30 30 2 30 328

Candida catenulata 1 2 3 6

C. intermedia 1 1

Clavispora lusitaniae 2 1 3

Rodothorula glutinis 1 1

Yarrowia lypolitica 1 1

Unidentified 20 20

Total 49 50 48 12 10 6 20 15 4 4 30 30 30 20 2 30 360

*Each number corresponds to a different worker.

CE, cheese at the early stage of ripening (4 days); CM, cheese at the middle stage of ripening (10 days); CL, cheese at the late stage of ripening

(16 days); WA, arms; WH, hands; WG, gloves; CR, air of the curing room; I, diluted inoculum; B1, 2 and 3, brine bath 1, 2 and 3; WS, wooden

shelves; SS, stainless shelves.

Sources of the adventitious microflora J. Mounier et al.

678 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681

ª 2006 The Authors

cessing and ripening in a Gouda factory but these workers

did not show whether the same strain was present on the

cheese surface and in the environment. In the present

study, the brines had high counts of this strain and,

therefore, are likely to be the major source of D. hansenii

for colonizing the young cheeses. This finding contrasts

with those of Petersen et al. (2002), who found that the

D. hansenii strains in the brine were not found on the

cheese surface after 3 days of ripening. This was probably

because of their slow growth on lactate. In our study, the

strain of D. hansenii in the brine microflora was able to

develop quickly on the cheese surface because a high pop-

ulation had been reached on day 4 postmanufacture or

2 days after the cheese was removed from the brine.

A progression of bacteria but not yeast was observed

during cheese ripening. Staphylococcus saprophyticus dom-

inated at day 4 and C. casei, C. variabile and Agrococcus

sp., the later stages of ripening (Fig. 2). This is in agree-

ment with previous findings on other smear cheeses

(Feurer et al. 2004; Rademaker et al. 2005) but it

contrasts with the previous study of Gubbeen where no

bacterial progression was observed (Brennan et al. 2002).

The ripening temperature and humidity are not con-

trolled during the ripening of this cheese, which may

explain these contradictory results.

The commercial ripening starters deliberately inocula-

ted onto the cheese surface were not recovered during

ripening except for one isolate of P. helvolus. Similar

results were obtained on a previous batch of Gubbeen

cheese (Brennan et al. 2002). In investigations on the

microflora of a German smear-ripened cheese, the bacter-

ial and yeast smear starters were also not detected at dif-

ferent stages of ripening (S. Goerges, J. Mounier, M. Rea,

R. Gelsomino, T.M. Cogan, J. Swings, M. Vancanneyt and

S. Scherer, unpublished data). Defined strain cultures are

being developed with some success (Bockelmann et al.

2000, 2005; Bockelmann and Hoppe-Seyler 2001). In the

present study, the ripening cultures were also not isolated

from the environmental samples or the skin of the work-

ers, except from the person who inoculated the cheeses

with the ripening starters. In addition, a lot of other

micro-organisms were isolated from the brine (Table 3)

or from the skin surface (Table 4), but they were not

reisolated from the cheese surface. However, some of

them have been isolated from the surface of other smear

cheeses, e.g. B. linens and A. arilaitensis from another

batch of Gubbeen cheese (Mounier et al. 2005), A. arilai-

tensis from Reblochon (Irlinger et al. 2005) and S. equo-

rum (Irlinger et al. 1997; Place et al. 2003). Several

hypotheses explain these findings. The organisms may be

present as subpopulations but are not part of the domin-

ant microflora. They may also be unable to grow under

the specific conditions of pH, salt and availability of

nutrients, on the cheese surface. Negative interactions

could have occurred between these organisms and the

dominant species in the cheese. Similarly, C. catenulata

and C. intermedia, which were isolated from the cheese

surface could not be reisolated in the cheese-making envi-

ronment and conversely, Clavispora lusitaniae, Rhodo-

torula glutinis and Yarrowia lipolytica isolated from the

skin, could not be reisolated from the cheese surface.

Except C. lusitaniae, all of these have been shown to be

minor components of the microflora of some cheeses, e.g.

C. catenulata, Y. lipolytica and R. glutinis on Gouda

cheese (Welthagen and Viljoen 1998), and C. catenulata,

C. intermedia and Y. lipolityca on Camembert and blue-

veined cheeses (Roostita and Fleet 1996). Other analytical

methods-like DGGE or TGGE could be useful to detect

bacterial and yeast subpopulations if they are present

(Ercolini et al. 2003; Meroth et al. 2003; Ogier et al.

2004).

Staphylococcus and Corynebacterium spp. are common

inhabitants of skin (Kloos and Musselwhite 1975; Noble

1984; Leyden et al. 1987) and are also common inhabit-

ants of the cheese microflora. This adds credence to the

idea that the skin and arms may be an important source

of these bacteria but this aspect was not studied in detail.

Handling the cheese would also affect transfer of these

organisms to the arms and hands of the workers. More

study is needed to establish if this, in fact, occurs. It is

not clear whether D. hansenii is an organism associated

with skin.

Overall, it is concluded that a progression of bacteria

but not yeast occurred during ripening and that the bac-

teria and the yeast developing on the cheese surface ori-

ginated from the cheese-making environment. This is the

first time that the direct involvement of the adventitious

yeast or bacterial microflora, from the cheese-making

environment, on the ripening of a smear-ripened cheese,

has been demonstrated. It was also shown that the yeast

and the staphylococci present on the cheese at an early

stage of ripening were identical to those present in the

brine. However, the source of the coryneforms that

develop later on the cheese surface remains unclear

although the skin of the workers could be a major source.

Nevertheless, it can be concluded that the skin is at least

a transient vector of these organisms.

Acknowledgements

J. Mounier thanks Teagasc for awarding him a Walsh

Fellowship. This study was partly financed by the Euro-

pean Community project ‘Biodiversity and anti-listerial

activity of surface microbial consortia from Limburger,

Reblochon, Livarot, Tilsit and Gubbeen cheeses’, QLK1-

2001-O2228.

J. Mounier et al. Sources of the adventitious microflora

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 668–681 679

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