International Journal of Cosmetic Science, 2014, 36, 386–395

doi: 10.1111/ics.12140

Enterobacter gergoviae adaptation to preservatives commonly used in cosmetic industry M. Periame, J.-M. Pages and A. Davin-Regli UMR-MD-1, Aix-Marseille Universite, IRBA, Transporteurs Membranaires, Chimioresistance et Drug Design, Marseille, France

Received 21 November 2013, Accepted 26 April 2014

Keywords: cosmetics, Enterobacter gergoviae, genetic analysis, microbiology, preservatives, safety testing

Synopsis OBJECTIVE: The aim of this study was to obtain a better understanding regarding the origin of recurrent contamination by Enterobacter gergoviae in diverse cosmetic formula. We studied 65 isolates collected from various sources (clinical, food, cosmetics). METHODS: RAPD analysis using AP12H, REP and ERIC-PCR was carried out for epidemiological typing. Evaluation of susceptibility to preservatives currently used in cosmetics for a representative panel of collection strains was measured. Preservative efficacy was evaluated by minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs). RESULTS: Eighty per cent of isolates was unrelated. E. gergoviae showed significant levels of resistance to preservatives. MBC was higher than maximum permitted concentrations imposed by European Commission (EC). Association of preservatives showed in rare case additive effects, and no synergic effects were observed. CONCLUSION: Most of the cosmetic formulations are contaminated with unrelated E. gergoviae strains. Maximum allowed concentrations for sodium benzoate are inefficient to limit proliferation and control adaptability to this bacterium in cosmetic products. Efflux mechanisms should be involved in methylisothiazolinone– chloromethylisothiazolinone and triclosan adaptation.  sume  Re OBJECTIFS: La finalite de ce travail est de determiner les facteurs responsables des contaminations recurrentes de cosmetiques de natures diverses, par E. gergoviae. Provenant de sources variees, 65 souches de E. gergoviae ont ete etudiees. METHODES: Une caracterisation genotypique a ete menee par marquage epidemiologique par RAPD. La sensibilite aux conservateurs antibacteriens les plus couramment utilises dans le domaine cosmetique a ete evaluee par des tests de CMIs et CMBs. L’effet additif ou synergique de leur association dans les formulations a ete egalement etudie. RESULTATS: La variabilite genetique de E. gergoviae est importante. 80% des isolats ne sont pas relies entre eux, par contre 3 episodes de contaminations par un m^eme isolat sur des periodes de 2 a 4 mois ont pu ^etre mis en evidence dans une m^eme entreprise et associes  a la persistance de la souche. Les CMBs sont atteintes pour des concentrations plus elevees que les concentrations d’usage Correspondence: Anne Davin-Regli, UMR-MD1, Transporteurs Membranaires, Chimioresistance et Drug Design, Facultes de Medecine et Pharmacie, 27 Bd Jean Moulin, 13385 Marseille cedex 05, France. Tel.: +33491835695; fax: +33491324606; e-mail: anne-veronique. [email protected]


autorisees par la Commission Europeenne. L’association des conservateurs entre eux montre uniquement des effets additifs sur l’inhibition de la croissance bacterienne mais pas d’effet synergique. CONCLUSIONS: La contamination des produits cosmetiques se fait  partir d’une source nouvelle, par des isolats differle plus souvent a ents. Cependant, la persistance de la bacterie au sein d’une m^eme cha^ıne de fabrication, a pu expliquer la contamination de lots de produits differents. Les tests effectues en presence d’un inhibiteur de l’efflux montrent que ce mecanisme n’est pas implique dans l’adaptation aux conservateurs etudies, sauf pour deux d’entre eux (le Triclosan et le melange methylisothiazolinone et chloromethylisothiazolone). Enfin, le benzoate de sodium est inefficace, pour emp^echer la proliferation de E. gergoviae, aux concentrations autorisees par la Commission Europeenne. Introduction Enterobacter gergoviae is reported in diverse human infectious diseases and under-represented in the collection of hospital laboratories [1–5]. The few available studies have reported that isolated strains were generally susceptible to antibiotics [1, 6, 7]. Enterobacter gergoviae has been isolated from many plants: maize [8], grape bay [9], various vegetable [7], coffee beans [10] and spring water [11]. It was also found in insect gut [12] such as Anastrepha [13], more commonly known as fruit flies and also in the gut of the pink bollworm and Pectinophora gossypiella, known for being a pest in cotton farming [14]. Adult Anastrepha food is fruit juice [13]. This insect regurgitates food on leaf surface. So fruit fly inoculated leaves and use bacterial colonies as a source of protein [12]. An environmental biotope is suggested and more specifically a vegetal environment. Interestingly, E. gergoviae is often identified by the quality control laboratories in contaminated recent manufactured or spoiled cosmetic products [6, 15, 16]. It has been shown that E. gergoviae exhibited an innate resistance to parabens, due to the production of an enzyme PrbA and an efflux mechanism [16]. Parabens, methylparaben and propylparaben the most common of these, stay preservatives used in cosmetic and personal care products, despite the controversial reputation and decrease in their use in cosmetic formulations [17]. Consequently, use of other antimicrobial agents such as phenols, organic and inorganic acids: esters and salts (dehydroacetic acid and levulinic acid) and preservative miscellaneous such as isothiazolinones and phenoxyethanol is preferred [18], increasing their delivery and activity, for efficiently limit the microbial growth [19–22]. In contrast to the various antibiotic families, preservatives and other antibacterial agents have

© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie

Preservatives and E. gergoviae adaptation

generally not only one specific microbial target [23], for example, low concentrations of triclosan inhibit synthesis of the key enzyme Fab I involved in fatty acid biosynthesis, and high concentrations also disrupt the membrane and precipitates the cytoplasmic compounds [24–28]. Membrane-active microbicides directly affect outer and/or inner membranes by a lytic effect or an ionophoric interaction [29–32]. Despite the great diversity of preservative targets, the contamination frequency of cosmetics by E. gergoviae is recurrent and very few and sparse information was disclosed by cosmetic companies about the origins and factors involved during contamination events [6, 15]. It is important for the cosmetic industry to have a better understanding of the adaptive mechanisms of E. gergoviae to preservatives used in cosmetics, to prevent the emergence and spreading of resistant strains. The objectives were to identify origins of the cosmetic contaminations and the mechanisms involved in preservative resistance using the strains previously described [6] and strains obtained from a cosmetic manufacturer (Table I). Cosmetics concerned by the E. gergoviae contaminations are often made of plant extracts such as verbena, lavender, Angelica, orange and Helichrysum. To identify the origin of the strain causing contamination of cosmetics, research of E. gergoviae was performed on the corresponding plants, factory environment, raw material plants and process water, without success. We determined the strain susceptibilities towards the various preservatives currently used in cosmetic formulations alone or in combination ( (Table II). The possible involvement of efflux mechanisms in bacterial adaptation towards various biocides is investigated using the efflux inhibitor phenylalanine arginine b-naphthylamide (PAbN), as previously reported [33–35]. Efflux pumps confer resistance to a wide range of compounds such as antibiotics, detergents and biocides, leading to potential risks associated with the preservative misuse/overuse and the selection of antibiotic-resistant bacteria [36–40]. For comparison, we studied triclosan (Irgasan), a well-known preservative studied for its mechanism of action, bacteria susceptibility or resistance [29, 41, 42]. This study demonstrated the genetic diversity of strains involved in cosmetic contaminations and the emergence of well-adapted bacteria demonstrating increase tolerance to one or more preservatives. Materials and methods Bacterial strains and growth conditions A total of 67 bacterial strains were collected in this study and are listed in Table I: E. gergoviae reference strain CIP 76.01 and six clinical isolates, 48 E. gergoviae isolated from various formulations of cosmetics (shampoos, creams, gel, water, eye liner, foam), five of them were studied in a precedent study (eg1-10-14-18-21) [6], nine isolated from kitchens surfaces, food or vegetables [6, 7], and one cosmetic isolate transformed with plasmid P9 including gene coding for MarA [6] and reference strains (E. aerogenes ATCC13048, Escherichia coli AG100). Isolates were checked to be E. gergoviae by the API 20 E system or VITEKâ 2-AST N017 identification card  (bioMerieux, Marcy-l0 Etoile, France). The Mueller-Hinton (MH) medium was used for preservative and solvent susceptibility tests. Epidemiological typing The isolates were investigated by RAPD with primers AP12H, REP and ERIC-PCR primer ERIC2 described previously [6, 43].

M. Periame et al.

Fingerprint patterns were compared without knowledge of epidemiologic data. Heterogeneity due to the intensities and the shapes of bands was not considered to be a difference. Strains were considered different when their profiles differed by two or more bands according to previous studies [6]. For the PCR-based techniques used, reproducibility was determined by testing independent DNA preparations extracted from cultures of single colonies at different times and amplified independently. Preservative and solvent susceptibility testing A panel of preservatives belonging to different chemical classes was used. Compounds’ trade names and providers were as follows: polyaminopropyl biguanide: CosmocilTM CQ (Arch); methylisothiazolinone (0.01%): NeoloneTM 950 (Dow Personal Care); methylisothiazolinone and chloromethylisothiazolinone (MIT/CMIT) 0.0015%: KathonTM CG (Dow Personal Care); pentylene glycol (1–5%): Hydroliteâ 5 (Symrise, Holzminden, Germany); levulinic acid (0.3–1%): Dermosoftâ 700B (Dr Staetmans); phenoxyethanol (1%): SepicideTM LD (Seppic); 5-chloro-2-(2,4-dichlorophenoxy) phenol (0.3% = 3 mg mL1): Triclosan (Sigma, Saint-Louis, MO, U.S.A.). Solvent concentrations used and maximum permitted concentrations allowed by the European Commission (EC) regulations are listed in Table II. Minimum inhibitory concentrations and MBC determination The minimum inhibitory concentrations (MICs) were determined on a panel of 12 strains illustrating the diverse origins of the collection, using 96-well plates. A concentration range was established for each preservative (Table II). Preservatives are prepared using a standard two-fold dilution method in MH medium [44]. Wells containing 100 lL of preservative dilution twice concentrated were inoculated with 100 lL of cell suspension prepared by diluting a culture in MH twice concentrated to obtain viable counts of approximately 105 colony-forming unit (CFU) mL1 [45] (Table II). The microplates were incubated at 30°C. After overnight incubation, MIC was measured and susceptibility to antibiotics was also tested using the same method [44]. The pH value was measured for each preservative dilution. Solvents were used at subinhibitory concentrations that not interfere with the effect of the compounds tested. The MICs for preservatives were determined in the presence of the efflux pump inhibitor PAbN, used at a final concentration of 26 mg L1 [6, 33, 35]. The minimum bactericidal concentration (MBC) was determined using two methods. The first method consisted of determining MBC after carrying out the MIC in a final volume of 1 mL: samples for which no visible bacterial growth was observed were centrifuged at 5000 g, 15 min at 20°C, pellets were resuspended in 100 lL Luria-Bertani fresh medium, and 60 lL was plated on Luria-Bertani agar. In the second method, MICs of preservatives were determined in 96-well plates. Bacterial suspensions in well plates were resuspended at 1/10e and 1/100e in Eugon LT100 (Fischer Scientific Bioblock, Illkirch, France) (neutralizing solution commonly used in cosmetic laboratories to check the presence of surviving bacteria by inhibiting preservative activity). One millilitre was spread on LuriaBertani agar plates. The MBC was defined as the lowest bactericidal concentration of preservatives (required to kill bacteria after incubation at 30°C for 48 h). Assays were repeated three times for

© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 36, 386–395


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Table I Bacterial strains used in this study, strains eg1-10-14-18-21 provided in a precedent study: [6], eg53 to eg56 come from K. Schwaiger: [7]

Bacterial strains

Enterobacter gergoviae CIP 76.01 (CDC604-77) CIP 76.02 (CDC605-77) CIP 79.51 CIP 104955 (CDC25-75) CIP 104981 ATCC 33426 ATCC33428

CIP 105140

Major characteristics


Name in this study

IP, Urine, France IP, Urine, France Sputum PAD PAD2-78 Human abscess Human sputum Cream Exfoliating gel Eye liner Foundation cream Foundation cream Shampoo Water rinsing Cream Shampooing Cream Exfoliating gel Cream Dietary supplement Syrup Cleansing lotion Rose bath Cream Cream Shower gel Shea butter Shea butter Foam Cream Foam Foam Foam Foam Countertop for plant Countertop for plant Countertop for plant Foam Foam Shampoo Shampoo Conditioner Shampoo Cleansing gel Cosmetic water Bubble bath Cleaning solution Shower gel Shampoo Foam Cosmetic water

Brenner et al. 1980 Brenner et al. 1980 Brenner et al. 1980 Brenner et al. 1980 Brenner et al. 1980 Brenner et al. 1980 Brenner et al. 1980 Cosmetic Industry1, 1992 Cosmetic Industry1, 1996 Cosmetic Industry1, 1997 Cosmetic Industry1, 1998 Cosmetic Industry1, 1998 Cosmetic Industry1, 1999 Yves Rocher Lab, France (challenge test), 1998 Cosmetic Industry2, 1999 Cosmetic Industry2, 1999 Cosmetic Industry2, 1999 Cosmetic Industry2 Cosmetic Industry2 Food Industry, Canada Food Industry, Canada Cosmetic Industry1, 1998 Cosmetic Industry3, 2009 Cosmetic Industry3, 2009 Cosmetic Industry3, 2009 Cosmetic Industry3, 2009 1999 1999 Cosmetic Industry3, 2010 Cosmetic Industry3, 2010 Cosmetic Industry3, 2010 Cosmetic Industry3, 2010 Cosmetic Industry3, 2010 Cosmetic Industry3, 2010 Hospital kitchen, Aubagne, 2011 Hospital kitchen, Aubagne, 2011 Hospital kitchen, Aubagne, 2011 Cosmetic Industry3, 2010 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 Cosmetic Industry3, 2011 K. Schwaiger University of Munich K. Schwaiger University of Munich K. Schwaiger University of Munich K. Schwaiger University of Munich Cosmetic Industry3, 2012 Cosmetic Industry3, 2012 Cosmetic Industry3, 2012 Cosmetic Industry3, 2012 Cosmetic Industry3, 2012 Cosmetic Industry3, 2012 Cosmetic Industry3, 2012 Cosmetic Industry3, 2012

eg1 eg2 eg3 eg4 eg5 eg6 eg7 eg8 eg9 eg10 eg11 eg12 eg13 eg14 eg15 eg16 eg17 eg18 eg19 eg20 eg21 eg22 eg23 eg24 eg25 eg26 eg27 eg28 eg29 eg30 eg31 eg32 eg33 eg34 eg35 eg36 eg37 eg38 eg39 eg40 eg41 eg42 eg43 eg44 eg45 eg46 eg47 eg48 eg49 eg50 eg51 eg52 eg53 eg54 eg55 eg56 eg57 eg58 eg59 eg60 eg61 eg62 eg63 eg64

Food Food Food Food

Shampoo Shampoo Cosmetic water Cosmetic water Cosmetic water Cosmetic water


© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 36, 386–395

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Table 1 (continued) Bacterial strains

Escherichia coli AG100 Enterobacter aerogenes ATCC 13048

Major characteristics


Name in this study

Foundation cream

Cosmetic Industry1, 1998 transformed with plasmid P9 including gene coding for MarA Protein (Davin-Regli et al. 51)


Wild-type E. coli K-12

Viveiros, 2005


a, 1998 Malle


each compound. In this study, MBC was estimated as the lowest concentration of preservative without visual turbidity using well plates method and the absence of colony in the case of agar plates. Combination of preservatives To determine the effects of preservatives combination, we used the synergy test protocol previously described [21] with some modifications: sterile strips (paper 5 mm 9 50 mm) were saturated with pure solutions of preservatives. After drying, saturated strips were applied on MH agar plates. Strip diffusion tests were each performed in duplicate. All plates were incubated overnight at 30°C. Associative effects (additive, synergic, antagonist and indifferent) were checked from the growing colony in diffusion zone of the two compounds. Results Epidemiological typing AP12H, REP and ERIC-PCR with single primers successfully typed all the E. gergoviae isolates. Patterns obtained with independent extracts from one isolate and amplified in two independent experiments exhibited equivalent patterns except for intensity level (data not shown). RAPD and ERIC fingerprint interpretation was correlated between the two PCR-typing methods. The results from the PCR-typing methods for strains isolated from the same cosmetic industry are presented in Fig. 1. Most of clinical isolates, strains used as controls and strains obtained from kitchens or food and those studied in a precedent study generated different patterns, demonstrating the epidemiological diversity of the species [6]. About the strains from the same cosmetic industry, we observed that during the four-year period (2009–2012), most of them were epidemiologically unrelated, but three episodes of contaminated products (March–April 2011, September–November 2011 and January–April 2012) due to a same strain associated with each event could be identified. With the exception of these three clusters of contaminations associated in the time to an indistinguishable isolate, the 53 other isolates were unrelated, suggesting that E. gergoviae is a ubiquitous species and that sources of contamination are diverse. Preservative susceptibility and effect of PAbN on MICs and MBCs We chose MIC and MBC methods, in conditions similar to what is done with antibiotics, because they allow the rapid evidence of bacterial resistance in response to biocide contact and action in general and to preservatives in particular. Even if the challenge test is

the validated test for evaluating the effectiveness of a biocide in the conditions of use, it is not suitable for precisely studying the mechanisms of bacterial resistance. Susceptibility of six preservatives and two stabilizers was tested on a panel of 12 strains of the diverse origins (Table III). The pH measured was of 3 for MIT/CMIT; 5 for sodium benzoate, methylisothiazolinone, pentylene glycol, levulinic acid and phenoxyethanol and 7 for polyaminopropyl biguanide and triclosan. Minimum inhibitory concentration values obtained using these collections (strains and products) are included in the levels corresponding to maximum permitted concentrations (Table II) for most products. It is worthwhile to note that in the case of sodium benzoate, nine strains exhibited a MIC above the maximum permitted concentrations. Regarding the MBCs obtained by two methods, the two approaches indicated that MBC values were higher than those of MIC for all strains except for pentylene glycol and phenoxyethanol that for some strain MBC are equal to MIC (Tables III and IV). Furthermore, MBCs were – higher than maximum permitted concentrations with the two methods used for pentylene glycol (except strains eg24 and eg25), MIT and levulinic acid – included maximum permitted concentrations for polyaminopropyl biguanide, phenoxyethanol and MIT + CMIT, excepted for strains eg24, eg29 and ea1 (Table IV). But in the case of neutralization of phenoxyethanol by dilution at 1/100e, MBC was higher for the strain eg10-14-21. MBCs obtained for triclosan were bellow the maximum permitted; however, concentration of triclosan superior to 20 lg mL1 could not be tested due to solubilization problems. Interestingly, the addition of PAbN, a blocker of efflux pump activity, showed a significant effect on MIC values for MIT + CMIT and triclosan: we observed a decrease in MIC of at least 4–7 times for triclosan and 2–6 times for MIT + CMIT, respectively. This potentialization suggests the involvement of an active efflux susceptible to PAßN in the E. gergoviae tolerance towards these preservatives (Table III). Effect of preservative combination The combination of two preservatives was measured by the use of sterile strips saturated with pure solution of each compound. An additive effect was observed when no bacterial growth was detected on the junction angle of two strips (each strip being soaked with a single product). The phenoxyethanol demonstrated additive effects with polyaminopropyl biguanide and levulinic acid (data not shown). Moreover, MIT has brought additive effects with levulinic acid. However, no synergic effect was noted with the various molecules (noticeable inhibition of bacterial growth in the junction area).

© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 36, 386–395


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Table II Compounds used in this study

Range tested

[Mother solution] – [solvents]

Sodium benzoate (rinse-off, 2.5%; oral, 1.7%; leave-on, 0.5%)*



Polyaminopropyl biguanide (0.3%)*

3 9 103–0.05%


Methylisothiazolinone (0.01%)*

5 9 103–0.32%

0.64 %

Methylisothiazolinone and chloromethylisothiazolinone (MIT/CMIT 0.0015%)*

2.92 9 105–7.5 9 104%


Stabilizers Pentylene glycol (1–5%)*



Levulinic acid (0.3–1% at pH < 5.5)*

7.5 9 102–4.8%


Preservatives solubilized in methanol Phenoxyethanol (1%)*

1.4 9 102–0.9%


0.15–10 µg mL1

20 µg mL1–0.1%

Solvents Methanol (Fischer scientific)



Dimethyl sulfoxide (DMSO) (Sigma-Aldrich)



NaOH (Merck)







Chemical structures

Preservatives solubilized in water

Preservatives solubilized in DMSO 5-chloro-2-(2,4-dichlorophenoxy)phenol (0.3% = 3 mg mL1)*


*Maximum permitted concentrations.


© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 36, 386–395

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59 60

strains detected

time period of contaminations































06sept 2009

11sept 2010

11-oct 2010

12-oct 2010

01-nov 2011

02-nov 2011

03-mar 2011

04-apr 2011

09-sep 2011

11-nov 2011

01-jan 2012

04-apr 2012

Figure 1 Epidemiological typing after RAPD and ERIC-PCR profile analysis interpretation for the E. gergoviae isolates collected by the cosmetic industry 3 from their controls, during the four-year period. White rectangles correspond to unrelated strains and rectangles with a same colour correspond to an identical strain associated with one episode involving various contaminated products by a same strain.

Discussion E. gergoviae is a Gram-negative bacteria involved in recurrent events of withdrawal of cosmetic products as the second Gram-negative bacteria at with Pseudomonas aeruginosa after Burkholderia cepacia, which has the highest prevalence in a FDA study on recall sterile products [6, 15, 46–48]. The aim of this study was to identify the origin of the contamination by E. gergoviae strain and understand how this strain is suitable for cosmetic environments

by studying the susceptibility to different preservatives commonly used. Detection assays of E. gergoviae were performed on the corresponding plants, whose extracts entered into the composition of contaminated cosmetics, process water and raw materials without success. Origin of contaminations of cosmetics by this species is unknown, and no specific sources could be identified. RAPD analysis has demonstrated that 80% of isolated strains were unrelated, suggesting that this bacterium is ubiquitous in the environment and a large variation in sources of contamination. The remaining

Table III MICs obtained without and with PAbN (26 mg L1) in grey columns, from 10 preservatives and stabilizers and four solvents. They were realized on the 10 E. gergoviae selected and the reference strains of Escherichia coli (ec1) and Enterobacter aerogenes (ea1)


Preservatives and stabilizers


Sodium benzoate (%) (rinse-off, 2.5%; oral, 1.7%; leave-on, 0.5%)*

Sodium benzoate (%) + PAbN

Polyaminopropyl biguanide (%) (0.3%)*

eg1 eg10 eg14 eg18 eg21 eg23 eg24 eg25 eg26 eg65 ec1 ea1

1.6 3.2 3.2 3.2 3.2 3.2 3.2 3.2 1.6 3.2 3.2 1.6

1.6 3.2 3.2 3.2 3.2 3.2 3.2 3.2 1.6 nd nd nd

3.9 3.9 3.9 3.9 3.9 7.8 3.9 7.8 7.8 0.0125 >0.0125 >0.0125 >0.0125 0.0125 0.0125 >0.0125 >0.0125 0.00625 >0.0125 >0.0125 >0.0125

>0.32 0.32 >0.32 0.08 0.16 0.16 >0.32 0.16 >0.32 0.16 >0.32 >0.32



1.875 E4 1.875 E4 1.875 E4 9.375 E5 3.75 E4 0.0015 >0.0015 9.375 E5 0.0015 >0.0015 7.5 E5 >0.0015


3.75 E4 1.875 E4 1.875 E4 1.875 E4 1.875 E4 0.0015 7.5 E4 9.375 E5 0.0015 >0.0015 3.75 E4 >0.0015

MBC, Minimum bactericidal concentration. *Correspond to values of maximum permitted concentration imposed by the European Commission (EC).








(%) (0.0015%)*


Polyaminopropyl biguanide

Minimal bactericidal concentration

6.4 6.4 6.4 3.2 6.4 6.4 3.2 3.2 6.4 12.8 6.4 6.4



glycol (%)


6.4 6.4 6.4 6.4 6.4 6.4 3.2 3.2 6.4 6.4 6.4 6.4



2.4 4.8 2.4 4.8 2.4 4.8 >4.8 2.4 1.2 2.4 2.4 2.4

>4.8 >4.8 4.8 4.8 2.4 >4.8 >4.8 2.4 2.4 2.4 2.4 2.4

0.9 0.9 0.9 0.9 0.9 0.9 0.45 0.45 0.9 0.9 0.9 0.9


0.9 1.8 1.8 0.9 1.8 0.9 0.45 0.45 0.9 0.9 0.9 0.9



pH < 5.5)*


Phenoxyethanol (%)

(0.3–1% at

acid (%)


Table IV MBCs obtained from six preservatives and stabilizers on the 10 E. gergoviae selected and the reference strains of Escherichia coli (ec1) and Enterobacter aerogenes (ea1)

>10 >10 10 10 >10 10 10 10 10 10 10 10


10 >10 >10 10 10 10 10 >10 10 10 10 10


(3 3 103 lg mL1)

phenol (µg mL1)



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Preservatives and E. gergoviae adaptation

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nation of different parameters dependent on the culture conditions as preservative concentration, incubation time, solubility of preservatives, use of bacterial active solvents, expression of mechanisms involved in preservative resistance, temperature, pH and agitation [39]. All these parameters can act positively or negatively on the bacterial growth in contrast to the bactericidal effect of preservatives and vice versa. Bacteria induce a pH increase in the medium, which also could probably decrease stability and/or efficacy of preservatives. Also, the use of the PAbN, an inhibitor of the efflux pumps, was associated with a significant decrease in the value of the MICs for MIT + CMIT and triclosan. These results indicate that a PAbN-sensitive mechanism can be involved in the E. gergoviae adaptability to growth in the presence of triclosan or MIT + CMIT

as seen elsewhere with E. coli, P. aeruginosa and E. aerogenes [35– 38, 49–51]. Different strategies could be employed by E. gergoviae face to preservatives, despite their use in combination, and explain its persistence in cosmetic products. Acknowledgements This work was partially supported by the Region PACA and Aix-Marseille University. We thank Jacqueline Chevalier and Jean-Michel Bolla for their fruitful discussions. This work was supported by the Aix-Marseille Universite and IRBA. This work was presented to the ICAAC 2013, September 10-13 in Denver, CO, USA.

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Enterobacter gergoviae adaptation to preservatives commonly used in cosmetic industry.

The aim of this study was to obtain a better understanding regarding the origin of recurrent contamination by Enterobacter gergoviae in diverse cosmet...
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