BACKGROUND
the surface of traditional smear-ripened cheeses is colonized by a complex microbial ecosystem.
its biodiversity has been investigated by identification of cultivable isolates with molecular techniques, such as pulsed-field gel electrophoresis , repetitive sequence-based pcr  and 16s rdna sequencing, or with fourier-transform infrared spectroscopy   <cit> .
biodiversity studies using culture independent fingerprinting techniques, such as temporal temperature gradient gel electrophoresis , denaturing gradient gel electrophoresis , single strand conformation polymorphism  and terminal restriction fragment length polymorphism , have revealed the presence of additional uncultivable species  <cit> .
the development of the smear is a dynamic process driven by metabiosis leading to the successive growth of several microbial communities.
the first microorganisms to colonize the surface are yeasts.
yeasts' deacidification properties create a favorable environment for the next populations, mainly staphylococci followed by coryneforms.
these two shifts in the microbial community structure of the smear have been observed in multiple studies  <cit> .
various marine bacteria have also been detected recently on cheese surface  <cit> .
population dynamics of complex cheese surface ecosystems at species level have been studied by cultivation methods, but these approaches are necessarily limited by the selectivity of the cultivation media chosen.
alternatively, fingerprinting techniques can be used to generate data on main populations of such ecosystems.
these methods are fast and can give a more exhaustive view of the biodiversity in cheese but they are greatly influenced by the quality of dna extraction protocols and bias may be introduced by the pcr amplification step  <cit> .
the main advantage of ttge/dgge over other available fingerprinting techniques is the possibility to identify detected species by sequencing of dna fragments after excision from the ttge/dgge gel  <cit> .
ttge/dgge has been applied to study dominant bacteria of dairy products, enabling detection of species accounting for at least  <dig> to 10% of the total flora, depending on the amplification efficiency of the pcr step for a given species  <cit> .
surface contamination of smear cheese by listeria monocytogenes is of concern for the industry since listeriosis breakouts have been associated with consumption of cheese  <cit> .
improvements in hygienic conditions and application of safety guidelines failed to reduce the contamination frequency to an acceptable level  <cit> .
growth of listeria on cheese surface is closely linked to the development of the surface ecosystems and is primarily supported by yeast growth, which leads to deacidification and provides nutrients for bacterial growth.
listeria sp.
has been shown to grow easily on smear cheeses when defined ripening cultures containing debaryomyces hansenii, geotrichum candidum and brevibacterium linens were used  <cit> .
certain complex consortia naturally developing on smear cheese surface have been shown to inhibit listeria sp.
in situ  <cit> .
in vitro studies of these anti-listerial activities led to the isolation of bacteriocin-producing strains among ripening microorganisms in certain cases  <cit> .
application of the bacteriocin producing strain on artificially contaminated cheeses failed however to fully restore the inhibition  <cit>  or disturbed the development of the smear  <cit> .
a better knowledge of microbial biodiversity and in situ population dynamics is crucial to identifying species that may be involved in the inhibition.
saubusse et al.
<cit>  successfully used this approach for detecting antilisterial flora naturally developing in the core of saint-nectaire type cheese.
the objective of the present study was therefore to investigate population dynamics of complex cheese surface consortia with respect to their in situ inhibition properties.
two surface consortia were isolated from commercial raclette type cheeses.
ttge was used for assessing biodiversity of both consortia at species level.
an in-house database for species-level identification of the bands appearing in the ttge fingerprints was developed with cultivable isolates.
the two complex consortia or a control flora  were then applied on freshly-produced raclette cheeses that were artificially contaminated with listeria innocua.
population dynamics and listeria growth were monitored over  <dig> to  <dig> ripening days.
RESULTS
bacterial biodiversity of cheese surface consortia by cultivation - development of a ttge profiles database
consortium f was serial plated on five selective and non-selective media.
a total of  <dig> cultivable isolates were subjected to ttge fingerprinting analysis and grouped into  <dig> ttge profiles.
one representative isolate of each profile was randomly selected and subjected to 16s rdna sequencing.
isolates displaying identical ttge profiles but isolated from different cultivation media or exhibiting different macroscopic or microscopic morphologies were also subjected to 16s rdna sequencing analyses.
the presence of  <dig> species was detected by cultivation, with  <dig> dominant species enumerated on tgya, the medium used for the determination of total cell count .
the number of bands and the corresponding migration lengths were recorded in a database .
a majority of species displayed ttge profiles with a single band for all isolates.
three species showed strain variations in ttge profiles, with some strains harboring  <dig> to  <dig> supplementary bands .
in addition, several species had indistinguishable ttge profiles.
profile  <dig> corresponded to both brachybacterium sp.
and arthrobacter arilaitensis, profile  <dig> to staphylococcus equorum, staphylococcus epidermidis and facklamia tabacinasalis, and profile  <dig> to both lactococcus lactis and marinilactibacillus psychrotolerans .
low-gc bacteria lc.
lactis and m. psychrotolerans could not be distinguished on low-gc gel whereas high-gc gel revealed specific bands for the two species .
the database  contained a total of  <dig> ttge profiles corresponding to  <dig> species.
it was used as reference for species-level identification in ttge fingerprints obtained by the culture independent approach.
brevibacterium linens
<dig>  <dig> isolates, i.e.
ca.
<dig> isolates per media, were analyzed by ttge and grouped into identical ttge profiles.
a representative isolate of each profile was identified by 16s rdna sequencing.
after the assignment of all isolates to a species, the percentage of each species on each of the five media was assessed.
the cell count of a species was calculated by multiplying percentage and cell count determined for the corresponding media.
<dig> closest 16s rdna sequence in the genbank public database http://www.ncbi.nlm.nih.gov.
<dig> total cell count was determined on tgya.
in addition, staphylococci were enumerated on bp agar and msa, lactic acid bacteria on mrs agar and enterococci on kfs agar.
<dig> given the polymorphy in the intraspecies diversity of b. linens , strain assignation to b. linens or the related species b. aurantiacum based on 16s rdna analysis only was considered not reliable.
bacterial biodiversity of cheese surface consortia by ttge fingerprinting
bacterial biodiversity of consortium f and m was assessed by ttge fingerprinting of total dna extracts, a culture independent method .
both consortia were analyzed on two gels, targeting the bacterial species with high-gc and low-gc content in separate runs.
ttge fingerprints of consortia f and m were composed of  <dig> bands each, corresponding to  <dig> and  <dig> species, respectively.
five bands could not be assigned to a known species of the database and were therefore submitted to cloning and sequencing after excision .
high similarity was found between consortium f and m with  <dig> common species, i.e.
corynebacterium variabile, microbacterium gubbeenense, an uncultured bacterium from marine sediment , corynebacterium casei, brevibacterium linens, staphylococcus equorum, lactococcus lactis, agrococcus casei and alkalibacterium kapii.
consortium f showed a higher diversity than consortium m with four additional species, brachybacterium tyrofermentans, brachybacterium sp., marinilactibacillus psychrotolerans and staphylococcus vitulinus.
the species brachybacterium paraconglomeratum was specific to consortium m.
<dig> these designations are used to annotate bands from ttge gels in figures  <dig> and  <dig>
<dig> closest 16s rdna sequence in the genbank public database http://www.ncbi.nlm.nih.gov.
3the 16s rdna sequence of band f exhibited highest similarity of 94% with clostridiisalibacter paucivorans , a bacterium that belong to cluster xii of the clostridium subphylum  <cit> .
population dynamics of cheese surface consortia by cultivation methods
total cell counts and yeast counts were similar for all cheeses, independent of the surface flora applied to cheeses, i.e.
consortium f, m or control flora omk  <dig>  total cell counts increased from  <dig>  ±  <dig>  ×  <dig> cfu cm- <dig> to  <dig>  ±  <dig>  ×  <dig> cfu cm- <dig> within  <dig> days and remained stable afterwards .
yeast counts increased from day  <dig> to reach  <dig>  ±  <dig>  ×  <dig> cfu cm- <dig> at day  <dig> and decreased afterwards by  <dig> to  <dig> log until the end of ripening.
mould counts of ca.
<dig> cfu cm- <dig> were measured after  <dig> weeks ripening on cheeses treated with consortium f, while no moulds were detected on the cheese treated with consortium m or on control cheese.
at the end of ripening, similar mould counts of ca.
<dig> cfu cm- <dig> were measured on all cheeses.
the ph of cheese surface increased from  <dig>  ±  <dig>  at day  <dig> to  <dig>  ±  <dig>  at day  <dig> to  <dig>  depending on the cheese, and was constant afterwards, with mean ph of  <dig>  ±  <dig> .
population dynamics of complex cheese surface consortia by ttge fingerprinting
population dynamics of consortium f or m were assessed at species level by ttge fingerprinting of total dna extracts .
ttge fingerprints of day  <dig> cheese depict the starter culture  as well as the composition of the smear brines.
multiple shifts in the microbial community structure of cheeses treated with complex surface consortia f or m were observed throughout ripening.
the nine species common to both consortia had similar sequential development on cheese surface.
lc.
lactis, used as starter culture for cheese manufacture, was part of the dominant flora until day  <dig> and disappeared thereafter.
st. equorum was the first species to colonize the surface within  <dig> days.
al.
kapii grew on day  <dig> concomitant with c. casei and b. linens, followed by c. variabile, an uncultured bacterium from marine sediment and mc.
gubbeenense between day  <dig> and  <dig>  agrococcus casei was first detected on day  <dig>  other species specific to consortium f  colonized the corresponding cheese after  <dig> to  <dig> days ripening.
both brachybacterium species also colonized the cheese treated with consortium m, but could only be detected after  <dig> days, together with the brachybacterium species specific to consortium m .
repetition of both treatments revealed the same trends with minor differences including a growth delay  for some high-gc species and the additional development of m. psychrotolerans at day  <dig> on the cheese treated with consortium m .
<dig> the letter  indicates sampling times where a given species was detected in the ttge gel.
the symbol  indicates growth of a species in the smear.
growth was assumed in two cases, i.e.
when a band was detected at a sampling time, while absent from the previous sampling time, or when the intensity of the detected band increased, compared to the previous sampling time .
2the same letter code as for band designation in figure  <dig> was used.
population dynamics of the defined commercial culture omk  <dig> by ttge fingerprinting
population dynamics of the defined commercial culture omk  <dig> at species level was assessed by ttge fingerprinting of total dna extracts .
all three species of the culture omk  <dig>  established themselves on cheese surface during the first  <dig> days.
each of the five b. linens strains of the culture omk  <dig> exhibited a distinguishable strain-specific ttge profile .
the profile of b. linens fam <dig>  was detected in the ttge fingerprint of day  <dig> cheese, showing that this strain predominated over other b. linens strains at the end of ripening.
additional species not deliberately applied on the cheese colonized the cheese surface along ripening.
two staphylococci species  appeared on day  <dig> as well as m. psychrotolerans and al.
kapii on day  <dig>  br.
tyrofermentans and an uncultured bacterium from marine sediment completed the high gc community at day  <dig>  repetition of the treatment revealed the same trends regarding the three defined species.
however, the development of non-deliberately applied species was different in the repetition.
three additional species colonized the cheese, i.e.
enterococcus sp., c. casei, ag.
casei, while br.
tyrofermentans could not be detected .
in situ inhibition of listeria by complex surface consortia
raclette cheeses were artificially contaminated with  <dig> strains of listeria innocua on day  <dig> and  <dig>  i.e.
when yeasts on cheese surface had reached high counts of  <dig>  ±  <dig>  ×  <dig> cfu cm- <dig>  from the amount added to the smear brine , listeria counts of  <dig>  ±  <dig>  ×  <dig> cfu cm- <dig>  and of  <dig>  ±  <dig>  ×  <dig> cfu cm- <dig>  were recovered from the surface immediately after contamination.
listeria development was strongly affected by the surface flora applied for ripening.
a decrease of listeria counts below the detection limit of the method  was observed for cheeses treated with complex consortia f or m supplemented with debaryomyces hansenii fam <dig> .
listeria could be recovered from cheese surface  with an enrichment procedure at the end of ripening , for both consortia.
in contrast, listeria counts on control cheeses treated with the commercial culture omk  <dig> increased to ca.
<dig> cfu cm- <dig> after one month .
discussion
to our knowledge, this work describes the first dynamic study of naturally developing anti-listerial cheese surface consortia.
the monitoring of two complex consortia obtained from industrial productions was carried out with ttge, a culture independent fingerprinting technique which enabled species-level detection of high-gc and low-gc bacteria in separate runs.
previous studies reported a broad range of biodiversity in smear consortia, with  <dig> to  <dig> bacterial species detected  <cit> .
high bacterial diversity was observed in consortium f, with  <dig> species detected at dominant level by culture independent analysis.
the cultivation approach detected only  <dig> of the  <dig> species present at dominant level in consortium f, but enabled detection of  <dig> additional species present at subdominant level.
ttge is a semiquantitative approach with limited sensitivity compared to the cultivation approach.
however, as fingerprinting technique, ttge enabled to overcome the arbitrary selection exercised on the flora by the cultivation step, giving a more complete view of biodiversity at dominant level.
the combined use of both approaches led to a detailed knowledge of biodiversity in cheese smear flora, as already observed by feurer et al.
and mounier et al.
<cit> .
the identification strategy used in the present study for the cultivation approach, i.e.
all cultivable isolates grouped by ttge profiles and subsequent sequencing, enabled the detection of intraspecies diversity differentiation in  <dig> dominant species.
this strategy greatly simplified the identification of bands in the ttge fingerprints of complex consortia corresponding to intraspecies variability.
consortium m displayed slightly less diversity than f with  <dig> species detected at the dominant level by culture independent analysis.
a total of  <dig> species were detected in consortia f and m, including eight coryneform bacteria.
c. variabile, c. casei, b. linens and mc.
gubbeenense are common ripening microorganisms of smear cheeses detected on soft cheeses  <cit>  and semi-hard cheeses  <cit> .
br.
tyrofermentans was first isolated from gruyère cheese  <cit>  and was recently shown to colonize the surface of soft cheeses  <cit> .
to our knowledge, this is the first time that br.
paraconglomeratum has been detected in cheese although this species has been previously isolated from milk  <cit> .
agrococcus casei was first isolated from gubbeen, an irish semi-hard cheese  <cit> .
three staphylococcus species were isolated in addition to coryneforms.
st. equorum is common on smear cheeses  <cit>  while st. vitulinus was only isolated by irlinger et al.
<cit>  from french cheeses.
st. epidermidis, a human skin inhabitant, was detected on various irish semi-hard cheeses  <cit> .
two gram-positive marine lactic acid bacteria  and an uncultured bacterium from marine sediment were also part of the dominant flora.
m. psychrotolerans has been detected in the smear of soft cheeses from germany and france  <cit> .
alkalibacterium sp.
was found to be present in many european cheeses including tilsiter, a semi-hard smear cheese  <cit> .
we also identified potentially undesirable species of enterococci in the subdominant flora of consortium f. enterococci have a controversial status in the dairy industry.
they are considered naturally occurring ripening organisms for artisan mediterranean cheese  <cit> , but also appear as emerging pathogens due to the virulence factors they tend to harbor  <cit> .
to our knowledge, this study is the first report of the presence of facklamia sp.
in cheese.
f. tabacinasalis was first isolated from powdered tobacco by collins et al.
<cit>  and has recently been detected in raw milk by delbès et al.
<cit>  in a french farm producing saint-nectaire cheese and by hantsis-zacharov and halpern  <cit>  in a farm from northern israel equipped with modern automated milking facilities.
the presence of f. tabacinasalis on the surface of smear cheese may constitute a health hazard, as this species was shown to be α-haemolytic on horse blood  <cit> .
moreover, from six facklamia species described to date, four were isolated from human clinical specimen  <cit> .
we observed highly similar microbial community structures of consortia f and m, with  <dig> species being common to both consortia at dominant level, despite different ripening procedures.
high interbatch diversity was described by rea et al.
<cit>  in a single cheese ripening facility of gubbeen, an irish semi-hard smear cheese, over  <dig> years production, which may be related to a lack of humidity and temperature control during ripening of gubbeen cheese.
in the present study, the production of swiss raclette type cheese with defined production and ripening parameters led to the development of a similar flora in two distinct dairies.
the source of this highly diverse flora remains unidentified but possible sources could be the brine bath, skin of the workers or wooden shelves, as shown by mounier et al.
<cit>  for gubbeen cheese.
the high biodiversity is particularly surprising in the case of dairy f, where the smear brine is freshly prepared prior to each smearing and inoculated with a defined ripening culture of only  <dig> bacterial species.
moreover, the smear brine is applied by a cheese ripening robot that smears the young cheeses first.
however, the microflora of the brine bath is not controlled and might be one of the major sources.
in particular, the brine bath  nacl) could be suitable to maintain the two halophilic and alkaliphilic marine lab detected in consortium f, as some strains of m. psychrotolerans and al.
kapii were shown to grow at salt concentration as high as 21%  by ishikawa et al.
<cit> .
dynamic studies of consortia f and m inoculated at same cell counts on cheese surface revealed a similar sequential development of nine bacterial species, i.e.
lc.
lactis, st. equorum, al.
kapii, c. casei, b. linens, c. variabile, an uncultured bacterium from marine sediment, mc.
gubbeenense and ag.
casei.
the development of this microbial community prevented growth of listeria innocua, inoculated at  <dig> ×  <dig> cfu ml- <dig> smear brine on cheeses at day  <dig> and  <dig>  over  <dig> to  <dig> days ripening.
contamination at day  <dig> and  <dig>  i.e.
when yeasts reached their highest density, provided optimal growth conditions for listeria, as shown by the rapid listeria growth on control cheese.
strong antilisterial activities were shown in this unfavorable condition for consortia f and m. antilisterial activities of complex undefined cheese surface consortia were already observed in previous studies  <cit> .
maoz et al.
<cit>  reported a total inhibition of l. monocytogenens during  <dig> days ripening of a soft smear cheese with an initial contamination level of  <dig>  ×  <dig> cfu ml- <dig> smear brine.
the surface of smear cheese contains a limited range of substrates supporting growth of microorganisms, mainly lactose and lactate.
lactose is mostly metabolized by lab during curd acidification and initial ripening.
the residual lactose can be metabolized on the cheese surface by yeasts during the first days of ripening, as shown for soft cheeses by leclercq-perlat et al.
<cit> .
lactate metabolized by yeasts into co <dig> and h2o leads to deacidification of the cheese surface  <cit> .
as a result, lactate continuously diffuses from the core to the surface of the cheese.
lactate can be totally consumed by surface microorganisms in soft cheeses  <cit> .
several smear bacterial species, i.e.
brevibacterium aurantiacum, c. casei, c. variabile, mc.
gubbeenense and st. saprophyticus, were shown to use lactate and casaminoacids for growth  <cit> .
in contrast, listeria sp.
can only use a limited range of carbon sources for growth, including glucose, glycerol, fructose and mannose, while no growth occurs on lactate or casaminoacids  <cit> .
premaratne et al.
<cit>  showed that listeria monocytogenes may utilize alternative carbon sources, such as n-acetylglucosamine and n-acetylmuramic acid, which are major components of bacterial and fungal cell walls  <cit> .
in addition, the yeast cell wall contains a mannan glycopeptide with mannose  <cit> , a sugar metabolized by listeria sp.
listeria growth on smear cheese can therefore be limited by a low availability of carbon source and stimulated by components of smear microorganisms.
marine lab ferment glucose into lactate and assimilate mannose  <cit> .
ishikawa et al.
<cit>  reported that al.
kapii can utilize a fairly limited range of carbon sources.
in the present study, m. psychrotolerans and/or al.
kapii established early on cheeses treated by complex consortia, i.e.
between day  <dig> and day  <dig>  we believe competition for nutrients between marine lab and listeria sp.
may be involved in listeria inhibition in the smear since the development of m. psychrotolerans and al.
kapii occurred simultaneously with the decrease of listeria counts for both cheeses treated with consortium f  and for one cheese treated with consortium m .
in addition, listeria growth on control cheese stopped when m. psychrotolerans and al.
kapii were first detected in the smear, i.e.
on day  <dig>  hain et al.
<cit>  reported a microarray experiment conducted with the antilisterial complex smear consortium described by maoz et al.
<cit> .
genes involved in energy supply were mostly up-regulated after  <dig> hours of contact between listeria monocytogenes and the consortium, suggesting that listeria had entered a state of starvation.
while maoz et al.
<cit>  detected m. psychrotolerans in the aforesaid smear consortium by cultivation methods, they may have overlooked the presence of al.
kapii or related species.
CONCLUSIONS
this work reports the first study of population dynamics of antilisterial cheese surface consortia.
dynamics of two consortia obtained from industrial productions revealed highly similar, with the sequential development of  <dig> common species, whereas development of both consortia inhibited listeria growth over the whole ripening period.
next to common cheese surface bacteria, the two consortia contained marine lactic acid bacteria  that developed early in ripening, shortly after the growth of staphylococci and concomitant with a decrease in listeria cell counts.
competition for nutrients between marine lab and listeria sp.
could be involved in the observed inhibition.
temporal temperature gradient gel electrophoresis revealed decisive to detect all marine bacteria present at dominant level in the smear, as only one of three species was detected by the culture dependent approach.
further cheese ripening experiments are needed to investigate the potential contribution of marine lab to antilisterial activity.

 the surface of traditional smear-ripened cheeses is colonized by a complex microbial ecosystem. its biodiversity has been investigated by identification of cultivable isolates with molecular techniques, such as pulsed-field gel electrophoresis, repetitive sequence-based pcr and 16s rdna sequencing, or with fourier-transform infrared spectroscopy. biodiversity studies using culture independent fingerprinting techniques, such as temporal temperature gradient gel electrophoresis, denaturing gradient gel electrophoresis, single strand conformation polymorphism and terminal restriction fragment length polymorphism, have revealed the presence of additional uncultivable species. the development of the smear is a dynamic process driven by metabiosis leading to the successive growth of several microbial communities. the first microorganisms to colonize the surface are yeasts. yeasts' deacidification properties create a favorable environment for the next populations, mainly staphylococci followed by coryneforms. these two shifts in the microbial community structure of the smear have been observed in multiple studies. various marine bacteria have also been detected recently on cheese surface. population dynamics of complex cheese surface ecosystems at species level have been studied by cultivation methods, but these approaches are necessarily limited by the selectivity of the cultivation media chosen. alternatively, fingerprinting techniques can be used to generate data on main populations of such ecosystems. these methods are fast and can give a more exhaustive view of the biodiversity in cheese but they are greatly influenced by the quality of dna extraction protocols and bias may be introduced by the pcr amplification step. the main advantage of ttge/dgge over other available fingerprinting techniques is the possibility to identify detected species by sequencing of dna fragments after excision from the ttge/dgge gel. ttge/dgge has been applied to study dominant bacteria of dairy products, enabling detection of species accounting for at least to 10% of the total flora, depending on the amplification efficiency of the pcr step for a given species. surface contamination of smear cheese by listeria monocytogenes is of concern for the industry since listeriosis breakouts have been associated with consumption of cheese. improvements in hygienic conditions and application of safety guidelines failed to reduce the contamination frequency to an acceptable level. growth of listeria on cheese surface is closely linked to the development of the surface ecosystems and is primarily supported by yeast growth, which leads to deacidification and provides nutrients for bacterial growth. listeria sp. has been shown to grow easily on smear cheeses when defined ripening cultures containing debaryomyces hansenii, geotrichum candidum and brevibacterium linens were used. certain complex consortia naturally developing on smear cheese surface have been shown to inhibit listeria sp. in situ. in vitro studies of these anti-listerial activities led to the isolation of bacteriocin-producing strains among ripening microorganisms in certain cases. application of the bacteriocin producing strain on artificially contaminated cheeses failed however to fully restore the inhibition or disturbed the development of the smear. a better knowledge of microbial biodiversity and in situ population dynamics is crucial to identifying species that may be involved in the inhibition. saubusse et al. successfully used this approach for detecting antilisterial flora naturally developing in the core of saint-nectaire type cheese. the objective of the present study was therefore to investigate population dynamics of complex cheese surface consortia with respect to their in situ inhibition properties. two surface consortia were isolated from commercial raclette type cheeses. ttge was used for assessing biodiversity of both consortia at species level. an in-house database for species-level identification of the bands appearing in the ttge fingerprints was developed with cultivable isolates. the two complex consortia or a control flora were then applied on freshly-produced raclette cheeses that were artificially contaminated with listeria innocua. population dynamics and listeria growth were monitored over to ripening days.  bacterial biodiversity of cheese surface consortia by cultivation - development of a ttge profiles database consortium f was serial plated on five selective and non-selective media. a total of cultivable isolates were subjected to ttge fingerprinting analysis and grouped into ttge profiles. one representative isolate of each profile was randomly selected and subjected to 16s rdna sequencing. isolates displaying identical ttge profiles but isolated from different cultivation media or exhibiting different macroscopic or microscopic morphologies were also subjected to 16s rdna sequencing analyses. the presence of species was detected by cultivation, with dominant species enumerated on tgya, the medium used for the determination of total cell count. the number of bands and the corresponding migration lengths were recorded in a database. a majority of species displayed ttge profiles with a single band for all isolates. three species showed strain variations in ttge profiles, with some strains harboring to supplementary bands. in addition, several species had indistinguishable ttge profiles. profile corresponded to both brachybacterium sp. and arthrobacter arilaitensis, profile to staphylococcus equorum, staphylococcus epidermidis and facklamia tabacinasalis, and profile to both lactococcus lactis and marinilactibacillus psychrotolerans. low-gc bacteria lc. lactis and m. psychrotolerans could not be distinguished on low-gc gel whereas high-gc gel revealed specific bands for the two species. the database contained a total of ttge profiles corresponding to species. it was used as reference for species-level identification in ttge fingerprints obtained by the culture independent approach. brevibacterium linens isolates, i.e. ca. isolates per media, were analyzed by ttge and grouped into identical ttge profiles. a representative isolate of each profile was identified by 16s rdna sequencing. after the assignment of all isolates to a species, the percentage of each species on each of the five media was assessed. the cell count of a species was calculated by multiplying percentage and cell count determined for the corresponding media. closest 16s rdna sequence in the genbank public database http://www.ncbi.nlm.nih.gov. total cell count was determined on tgya. in addition, staphylococci were enumerated on bp agar and msa, lactic acid bacteria on mrs agar and enterococci on kfs agar. given the polymorphy in the intraspecies diversity of b. linens, strain assignation to b. linens or the related species b. aurantiacum based on 16s rdna analysis only was considered not reliable. bacterial biodiversity of cheese surface consortia by ttge fingerprinting bacterial biodiversity of consortium f and m was assessed by ttge fingerprinting of total dna extracts, a culture independent method. both consortia were analyzed on two gels, targeting the bacterial species with high-gc and low-gc content in separate runs. ttge fingerprints of consortia f and m were composed of bands each, corresponding to and species, respectively. five bands could not be assigned to a known species of the database and were therefore submitted to cloning and sequencing after excision. high similarity was found between consortium f and m with common species, i.e. corynebacterium variabile, microbacterium gubbeenense, an uncultured bacterium from marine sediment, corynebacterium casei, brevibacterium linens, staphylococcus equorum, lactococcus lactis, agrococcus casei and alkalibacterium kapii. consortium f showed a higher diversity than consortium m with four additional species, brachybacterium tyrofermentans, brachybacterium sp., marinilactibacillus psychrotolerans and staphylococcus vitulinus. the species brachybacterium paraconglomeratum was specific to consortium m. these designations are used to annotate bands from ttge gels in figures and closest 16s rdna sequence in the genbank public database http://www.ncbi.nlm.nih.gov. 3the 16s rdna sequence of band f exhibited highest similarity of 94% with clostridiisalibacter paucivorans, a bacterium that belong to cluster xii of the clostridium subphylum. population dynamics of cheese surface consortia by cultivation methods total cell counts and yeast counts were similar for all cheeses, independent of the surface flora applied to cheeses, i.e. consortium f, m or control flora omk total cell counts increased from ± × cfu cm- to ± × cfu cm- within days and remained stable afterwards. yeast counts increased from day to reach ± × cfu cm- at day and decreased afterwards by to log until the end of ripening. mould counts of ca. cfu cm- were measured after weeks ripening on cheeses treated with consortium f, while no moulds were detected on the cheese treated with consortium m or on control cheese. at the end of ripening, similar mould counts of ca. cfu cm- were measured on all cheeses. the ph of cheese surface increased from ± at day to ± at day to depending on the cheese, and was constant afterwards, with mean ph of ±. population dynamics of complex cheese surface consortia by ttge fingerprinting population dynamics of consortium f or m were assessed at species level by ttge fingerprinting of total dna extracts. ttge fingerprints of day cheese depict the starter culture as well as the composition of the smear brines. multiple shifts in the microbial community structure of cheeses treated with complex surface consortia f or m were observed throughout ripening. the nine species common to both consortia had similar sequential development on cheese surface. lc. lactis, used as starter culture for cheese manufacture, was part of the dominant flora until day and disappeared thereafter. st. equorum was the first species to colonize the surface within days. al. kapii grew on day concomitant with c. casei and b. linens, followed by c. variabile, an uncultured bacterium from marine sediment and mc. gubbeenense between day and agrococcus casei was first detected on day other species specific to consortium f colonized the corresponding cheese after to days ripening. both brachybacterium species also colonized the cheese treated with consortium m, but could only be detected after days, together with the brachybacterium species specific to consortium m. repetition of both treatments revealed the same trends with minor differences including a growth delay for some high-gc species and the additional development of m. psychrotolerans at day on the cheese treated with consortium m. the letter indicates sampling times where a given species was detected in the ttge gel. the symbol indicates growth of a species in the smear. growth was assumed in two cases, i.e. when a band was detected at a sampling time, while absent from the previous sampling time, or when the intensity of the detected band increased, compared to the previous sampling time. 2the same letter code as for band designation in figure was used. population dynamics of the defined commercial culture omk by ttge fingerprinting population dynamics of the defined commercial culture omk at species level was assessed by ttge fingerprinting of total dna extracts. all three species of the culture omk established themselves on cheese surface during the first days. each of the five b. linens strains of the culture omk exhibited a distinguishable strain-specific ttge profile. the profile of b. linens fam was detected in the ttge fingerprint of day cheese, showing that this strain predominated over other b. linens strains at the end of ripening. additional species not deliberately applied on the cheese colonized the cheese surface along ripening. two staphylococci species appeared on day as well as m. psychrotolerans and al. kapii on day br. tyrofermentans and an uncultured bacterium from marine sediment completed the high gc community at day repetition of the treatment revealed the same trends regarding the three defined species. however, the development of non-deliberately applied species was different in the repetition. three additional species colonized the cheese, i.e. enterococcus sp., c. casei, ag. casei, while br. tyrofermentans could not be detected. in situ inhibition of listeria by complex surface consortia raclette cheeses were artificially contaminated with strains of listeria innocua on day and i.e. when yeasts on cheese surface had reached high counts of ± × cfu cm- from the amount added to the smear brine, listeria counts of ± × cfu cm- and of ± × cfu cm- were recovered from the surface immediately after contamination. listeria development was strongly affected by the surface flora applied for ripening. a decrease of listeria counts below the detection limit of the method was observed for cheeses treated with complex consortia f or m supplemented with debaryomyces hansenii fam. listeria could be recovered from cheese surface with an enrichment procedure at the end of ripening, for both consortia. in contrast, listeria counts on control cheeses treated with the commercial culture omk increased to ca. cfu cm- after one month. discussion to our knowledge, this work describes the first dynamic study of naturally developing anti-listerial cheese surface consortia. the monitoring of two complex consortia obtained from industrial productions was carried out with ttge, a culture independent fingerprinting technique which enabled species-level detection of high-gc and low-gc bacteria in separate runs. previous studies reported a broad range of biodiversity in smear consortia, with to bacterial species detected. high bacterial diversity was observed in consortium f, with species detected at dominant level by culture independent analysis. the cultivation approach detected only of the species present at dominant level in consortium f, but enabled detection of additional species present at subdominant level. ttge is a semiquantitative approach with limited sensitivity compared to the cultivation approach. however, as fingerprinting technique, ttge enabled to overcome the arbitrary selection exercised on the flora by the cultivation step, giving a more complete view of biodiversity at dominant level. the combined use of both approaches led to a detailed knowledge of biodiversity in cheese smear flora, as already observed by feurer et al. and mounier et al.. the identification strategy used in the present study for the cultivation approach, i.e. all cultivable isolates grouped by ttge profiles and subsequent sequencing, enabled the detection of intraspecies diversity differentiation in dominant species. this strategy greatly simplified the identification of bands in the ttge fingerprints of complex consortia corresponding to intraspecies variability. consortium m displayed slightly less diversity than f with species detected at the dominant level by culture independent analysis. a total of species were detected in consortia f and m, including eight coryneform bacteria. c. variabile, c. casei, b. linens and mc. gubbeenense are common ripening microorganisms of smear cheeses detected on soft cheeses and semi-hard cheeses. br. tyrofermentans was first isolated from gruyère cheese and was recently shown to colonize the surface of soft cheeses. to our knowledge, this is the first time that br. paraconglomeratum has been detected in cheese although this species has been previously isolated from milk. agrococcus casei was first isolated from gubbeen, an irish semi-hard cheese. three staphylococcus species were isolated in addition to coryneforms. st. equorum is common on smear cheeses while st. vitulinus was only isolated by irlinger et al. from french cheeses. st. epidermidis, a human skin inhabitant, was detected on various irish semi-hard cheeses. two gram-positive marine lactic acid bacteria and an uncultured bacterium from marine sediment were also part of the dominant flora. m. psychrotolerans has been detected in the smear of soft cheeses from germany and france. alkalibacterium sp. was found to be present in many european cheeses including tilsiter, a semi-hard smear cheese. we also identified potentially undesirable species of enterococci in the subdominant flora of consortium f. enterococci have a controversial status in the dairy industry. they are considered naturally occurring ripening organisms for artisan mediterranean cheese, but also appear as emerging pathogens due to the virulence factors they tend to harbor. to our knowledge, this study is the first report of the presence of facklamia sp. in cheese. f. tabacinasalis was first isolated from powdered tobacco by collins et al. and has recently been detected in raw milk by delbès et al. in a french farm producing saint-nectaire cheese and by hantsis-zacharov and halpern in a farm from northern israel equipped with modern automated milking facilities. the presence of f. tabacinasalis on the surface of smear cheese may constitute a health hazard, as this species was shown to be α-haemolytic on horse blood. moreover, from six facklamia species described to date, four were isolated from human clinical specimen. we observed highly similar microbial community structures of consortia f and m, with species being common to both consortia at dominant level, despite different ripening procedures. high interbatch diversity was described by rea et al. in a single cheese ripening facility of gubbeen, an irish semi-hard smear cheese, over years production, which may be related to a lack of humidity and temperature control during ripening of gubbeen cheese. in the present study, the production of swiss raclette type cheese with defined production and ripening parameters led to the development of a similar flora in two distinct dairies. the source of this highly diverse flora remains unidentified but possible sources could be the brine bath, skin of the workers or wooden shelves, as shown by mounier et al. for gubbeen cheese. the high biodiversity is particularly surprising in the case of dairy f, where the smear brine is freshly prepared prior to each smearing and inoculated with a defined ripening culture of only bacterial species. moreover, the smear brine is applied by a cheese ripening robot that smears the young cheeses first. however, the microflora of the brine bath is not controlled and might be one of the major sources. in particular, the brine bath nacl) could be suitable to maintain the two halophilic and alkaliphilic marine lab detected in consortium f, as some strains of m. psychrotolerans and al. kapii were shown to grow at salt concentration as high as 21% by ishikawa et al.. dynamic studies of consortia f and m inoculated at same cell counts on cheese surface revealed a similar sequential development of nine bacterial species, i.e. lc. lactis, st. equorum, al. kapii, c. casei, b. linens, c. variabile, an uncultured bacterium from marine sediment, mc. gubbeenense and ag. casei. the development of this microbial community prevented growth of listeria innocua, inoculated at × cfu ml- smear brine on cheeses at day and over to days ripening. contamination at day and i.e. when yeasts reached their highest density, provided optimal growth conditions for listeria, as shown by the rapid listeria growth on control cheese. strong antilisterial activities were shown in this unfavorable condition for consortia f and m. antilisterial activities of complex undefined cheese surface consortia were already observed in previous studies. maoz et al. reported a total inhibition of l. monocytogenens during days ripening of a soft smear cheese with an initial contamination level of × cfu ml- smear brine. the surface of smear cheese contains a limited range of substrates supporting growth of microorganisms, mainly lactose and lactate. lactose is mostly metabolized by lab during curd acidification and initial ripening. the residual lactose can be metabolized on the cheese surface by yeasts during the first days of ripening, as shown for soft cheeses by leclercq-perlat et al.. lactate metabolized by yeasts into co and h2o leads to deacidification of the cheese surface. as a result, lactate continuously diffuses from the core to the surface of the cheese. lactate can be totally consumed by surface microorganisms in soft cheeses. several smear bacterial species, i.e. brevibacterium aurantiacum, c. casei, c. variabile, mc. gubbeenense and st. saprophyticus, were shown to use lactate and casaminoacids for growth. in contrast, listeria sp. can only use a limited range of carbon sources for growth, including glucose, glycerol, fructose and mannose, while no growth occurs on lactate or casaminoacids. premaratne et al. showed that listeria monocytogenes may utilize alternative carbon sources, such as n-acetylglucosamine and n-acetylmuramic acid, which are major components of bacterial and fungal cell walls. in addition, the yeast cell wall contains a mannan glycopeptide with mannose, a sugar metabolized by listeria sp. listeria growth on smear cheese can therefore be limited by a low availability of carbon source and stimulated by components of smear microorganisms. marine lab ferment glucose into lactate and assimilate mannose. ishikawa et al. reported that al. kapii can utilize a fairly limited range of carbon sources. in the present study, m. psychrotolerans and/or al. kapii established early on cheeses treated by complex consortia, i.e. between day and day we believe competition for nutrients between marine lab and listeria sp. may be involved in listeria inhibition in the smear since the development of m. psychrotolerans and al. kapii occurred simultaneously with the decrease of listeria counts for both cheeses treated with consortium f and for one cheese treated with consortium m. in addition, listeria growth on control cheese stopped when m. psychrotolerans and al. kapii were first detected in the smear, i.e. on day hain et al. reported a microarray experiment conducted with the antilisterial complex smear consortium described by maoz et al.. genes involved in energy supply were mostly up-regulated after hours of contact between listeria monocytogenes and the consortium, suggesting that listeria had entered a state of starvation. while maoz et al. detected m. psychrotolerans in the aforesaid smear consortium by cultivation methods, they may have overlooked the presence of al. kapii or related species.  this work reports the first study of population dynamics of antilisterial cheese surface consortia. dynamics of two consortia obtained from industrial productions revealed highly similar, with the sequential development of common species, whereas development of both consortia inhibited listeria growth over the whole ripening period. next to common cheese surface bacteria, the two consortia contained marine lactic acid bacteria that developed early in ripening, shortly after the growth of staphylococci and concomitant with a decrease in listeria cell counts. competition for nutrients between marine lab and listeria sp. could be involved in the observed inhibition. temporal temperature gradient gel electrophoresis revealed decisive to detect all marine bacteria present at dominant level in the smear, as only one of three species was detected by the culture dependent approach. further cheese ripening experiments are needed to investigate the potential contribution of marine lab to antilisterial activity. 
ttge analyses revealed a similar sequential development of the nine species common to both consortia. surface contamination of smear cheese by listeria spp. ttge revealed nine bacterial species common to both f and m consortia, but consortium f exhibited a higher diversity than consortium m, with thirteen and ten species, respectively. a decrease of listeria counts was observed on cheese surface inoculated at day with - × cfu cm- when cheeses were smeared with consortium f or m. listeria counts went below the detection limit of the method between day and and no subsequent regrowth was detected over to ripening days. beside common cheese surface bacteria, the two consortia contained marine lactic acid bacteria that developed early in ripening, shortly after the growth of staphylococci. population dynamics were studied after application of the consortia on fresh-produced raclette cheeses. bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by temporal temperature gradient gel electrophoresis, a culture independent technique, for two microbial consortia isolated from commercial raclette type cheeses inoculated with defined commercial ripening cultures or produced with an old-young smearing process. this work reports the first population dynamics study of complex smear ecosystems exhibiting in situ antilisterial activity. in contrast, listeria grew to high counts on cheeses smeared with a defined surface culture. ttge revealed the presence of marine lactic acid bacteria that are likely related to the strong listeria inhibition, as their early development in the smear occurred simultaneously with a decrease in listeria cell count.
 surface contamination of smear cheese by listeria spp. is of major concern for the industry. complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by temporal temperature gradient gel electrophoresis, a culture independent technique, for two microbial consortia isolated from commercial raclette type cheeses inoculated with defined commercial ripening cultures or produced with an old-young smearing process.  ttge revealed nine bacterial species common to both f and m consortia, but consortium f exhibited a higher diversity than consortium m, with thirteen and ten species, respectively. population dynamics were studied after application of the consortia on fresh-produced raclette cheeses. ttge analyses revealed a similar sequential development of the nine species common to both consortia. beside common cheese surface bacteria, the two consortia contained marine lactic acid bacteria that developed early in ripening, shortly after the growth of staphylococci. a decrease of listeria counts was observed on cheese surface inoculated at day with - × cfu cm- when cheeses were smeared with consortium f or m. listeria counts went below the detection limit of the method between day and and no subsequent regrowth was detected over to ripening days. in contrast, listeria grew to high counts on cheeses smeared with a defined surface culture.  this work reports the first population dynamics study of complex smear ecosystems exhibiting in situ antilisterial activity. ttge revealed the presence of marine lactic acid bacteria that are likely related to the strong listeria inhibition, as their early development in the smear occurred simultaneously with a decrease in listeria cell count. 