Marina Périamé

Jean-Marie Pagès

Anne P Davin-Regli

Marina P Eriam

Jean-Marie Pag

Enterobacter gergoviae membrane modifications are involved in the adaptive response to preservatives used in cosmetic industry

Keywords: antibiotics and disinfectants cross-resistance, bacterial adaptation, cosmetics, Enterobacter gergoviae, preservatives, preservatives adaptive mechanisms

Enterobacter gergoviae membrane modifications are involved in the adaptive response to preservatives used in cosmetic industry

Introduction

Enterobacter gergoviae is a Gram-negative bacteria often identified in awry manufactured or spoiled cosmetic products [START_REF] Anelich | Survey of microorganisms associated with spoilage of cosmetic creams manufactured in south Africa[END_REF][START_REF] Chollet | Enterobacter gergoviae and the prevalence of efflux in parabens resistance[END_REF]. Recently, we have confirmed the genetic diversity of strains involved in cosmetic contaminations and the bacteriostatic action of preservatives which results in increased bacterial insusceptibility and thus its emergence (P eriam e et al. 2014). These days, to ensure microbiological quality, consumer safety and the organoleptic properties of cosmetic products, manufacturers need to comply with defined standards (BIO ECOCERT, COSMOS) [START_REF] Hiom | Preservation of medicines and cosmetics. Chapter 17[END_REF]). As a result, cosmetic production requires disinfection steps in the production chain before and during the manufacture/ conditioning stages. In this disinfection process, two parameters, concentration and contact time of preservative, play a key role, for example in case of methylisothiazolinone-chloromethylisothiazolinone (MIT-CMIT) [START_REF] Maillard | Factors affecting the activities of microbicides. Chapter 3[END_REF][START_REF] Maillard | Does microbicide use in consumer products promote antimicrobial resistance ? A critical review and recommendations for a cohesive approach to risk assessment[END_REF]. A drawback regarding the usage of these preservatives is the possibility to generate cross-insusceptibility to other disinfectants or preservatives among the microflora [START_REF] Maillard | Factors affecting the activities of microbicides. Chapter 3[END_REF][START_REF] Maillard | Does microbicide use in consumer products promote antimicrobial resistance ? A critical review and recommendations for a cohesive approach to risk assessment[END_REF]. Hence, understanding the molecular mechanism involved in the rise of this adaptation is necessary. This will lead to identification of novel drug targets and hence promote new modes of counteracting them. To attain this understanding, we have examined insusceptible strains derivatives from the susceptible Ent. gergoviae ATCC 33028, using increasing MIT-CMIT and triclosan concentrations [START_REF] Rensch | Analysis of triclosan-selected Salmonella enterica mutants of eight serovars revealed increased aminoglycoside susceptibility and reduced growth rates[END_REF]. These compounds are used in cosmetic industry for disinfection process. The resulting derivatives exhibited insusceptibility to biguanides and nonionic surfactants-amines which have a common mode of action on bacterial membranes [START_REF] Broxton | Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli[END_REF][START_REF] Ikeda | Interaction of a polymeric biguanide biocide with phospholipid membranes[END_REF][START_REF] Allen | The response of Escherichia coli to exposure to the biocide polyhexamethylene biguanide[END_REF][START_REF] Ortega Morente | Biocide tolerance in bacteria[END_REF]. The characteristics of these adapted strains were studied to determine the involvement of common mechanisms active in preservatives and in biocides insusceptibility. This point is of special importance with the European commission regulation concerning the use of biocidal products currently used in cosmetic formulations and the necessity to ensure that, when properly used, they are sufficiently effective and have no unacceptable effect on the target organisms such as resistance [START_REF] Anon | Enumeration and detection of aerobic mesophilic bacteria[END_REF].

Materials and methods

Bacterial strains, derivative strains and growth conditions

Enterobacter gergoviae clinical strain ATCC 33028, its biocide-resistant derivatives and three strains, namely eg23, eg25 and eg26, isolated from contaminated products from a cosmetic company (P eriam e et al. 2014) were used (Table 1). These three cosmetic strains were chosen for various preservatives susceptibility tests, out of a panel of 47 Ent. gergoviae isolated from cosmetic products (P eriam e et al. 2014). Bacteria were grown at 37°C in Luria-Bertani (LB) broth (Difco Laboratories, Detroit, MI, USA). Insusceptible derivate strains were successively obtained from ATCC 33028, which were grown in the presence of methylisothiazolinone, MIT-CMIT or triclosan, using culture with increased stepwise concentrations ranging from 1/2 MIC to 6 9 MIC of the selected compounds. Four MIT-CMIT insusceptible derivatives were obtained at 1Á875Á10 À4 %, 3Á75Á10 À4 %, 4Á5Á10 À4 % and 6Á10 À4 % concentrations and named M1, M2, M3 and M4, respectively. Similarly, three triclosan insusceptible derivatives were obtained at 5, 10 and 20 g l À1 concentrations, which were named T1, T2 and T3, respectively (Table 1). Mueller-Hinton broth (MHB) was used as the bacterial medium for preservatives, antibiotics and chemicals susceptibility tests.

Disinfectants susceptibility, MIC and MBC determination

Susceptibility to disinfectants was tested for peroxyacid (P3-oxonia), aminopropyl dodecylpropane-diamine combined with polyhexamethylene biguanide (ADD/PHMB) Bactanios2000 and laurylamine dipropylenediamine (Aniosteril DAC II). These actives are currently used in cosmetic industry during the production steps. The minimum inhibitory concentrations (MICs) were determined by the microdilution method (CLSI) in MHB in microplates as previously described [START_REF] Kuete | Antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype[END_REF]. MBCs were determined using Eugon LT100 broth in 96-well plates, which is commonly used in cosmetic industry for the enrichment of aerobic bacteria in cosmetic products and preservatives neutralization according to ISO standards [START_REF] Quisno | A neutralizing medium for evaluating the germicidal potency of the quaternary ammonium salts[END_REF]. The medium is composed of a mixture of peptones, cystine, glucose and salts which favour the growth of a wide variety of micro-organisms. Lecithin and polysorbate 80 neutralize the antibacterial activity of most antiseptics or preservatives [START_REF] Williamson | A new method for the quantitative investigation of cutaneous bacteria[END_REF].

Briefly, after MIC determination in MHB, the bacterial wells showing growth inhibition were resuspended at 1/10 and 1/100 in Eugon LT100 broth and re-cultivated. The MBC was defined as the lowest bactericidal concentration of preservatives required to kill bacteria after incubation at 30°C for one to 4 days as recommended for the enrichment of aerobic bacteria in cosmetic products according to ISO standards, respectively [START_REF] Anon | Enumeration and detection of aerobic mesophilic bacteria[END_REF]. The determinations were carried out in independent triplicates.

Antibiotics, preservatives and chemical susceptibility testing by MIC determination

Cefepime (CEF), nalidixic acid (NAL), polymyxin B (POL), ceftazidime (CAZ) and imipenem (IMI) were used to assess the antibiotic susceptibility of the various strains. Triclosan and MIT-CMIT were used to assess derivatives adaptation to these biocides compared to the reference strain and those isolated from cosmetic formulations. Antibiotics were prepared and used according to the microdilution method in MHB as previously described for disinfectants. Phenylalanine-arginine bnaphthylamide (PAßN) used at low concentrations has been previously reported to block the Enterobacteriaceae efflux pump such as AcrAB-TolC and restore the activity of antibiotic molecules that are well described as efflux pump substrates [START_REF] Mamelli | New antibiotic molecules: bypassing the membrane barrier of gram negative bacteria increases the activity of peptide deformylase inhibitors[END_REF][START_REF] Kuete | Antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype[END_REF]. PAßN was thus used as efflux pump inhibitor on the various Ent. gergoviae strains. The inoculated microplates were incubated at 30°C for 18-24 h. An efflux pump activity was identified when the PAbN addition induced a threefold decrease in MIC value for an antibiotic molecule [START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF]. Each assay was repeated independently three times, and the MIC values corresponded to the mean of these triplicates.

A way to circumvent alteration of bacterial membrane barrier in resistant strains is to use chaotropic agents or detergents [START_REF] Mamelli | New antibiotic molecules: bypassing the membrane barrier of gram negative bacteria increases the activity of peptide deformylase inhibitors[END_REF][START_REF] Bolla | Strategies for bypassing the membrane barrier in multidrug resistant Gram-negative bacteria[END_REF]. MICs determination of a chelating agent such as EDTA, which is used as a common food preservative or a detergent such as sodium deoxycholate (DOC), were employed to investigate bacterial outer membrane integrity of ATCC 33028 strain as compared to triclosan and MIT-CMIT derivative-resistant strains. The MIC assays were performed in 96-well plates. EDTA was prepared using a twofold dilution method in MHB. Wells containing 100 ll of EDTA dilution (29) were inoculated with 100 ll of cell suspension prepared by diluting a culture in double strength MHB (29) to obtain viable counts of approximately 10 5 CFU ml À1 . For combination assays, EDTA was used at 1 mmol final concentration as previously reported [START_REF] Mamelli | New antibiotic molecules: bypassing the membrane barrier of gram negative bacteria increases the activity of peptide deformylase inhibitors[END_REF].

b-Lactamase assays

The various bacterial cultures were incubated overnight in the presence of imipenem (at subinhibitory concentration) to induce the expression of ß-lactamase [START_REF] Fadli | Thymus maroccanus essential oil, a membranotropic compound active on Gramnegative bacteria and resistant isolates[END_REF] with the respective concentrations of preservatives (MIT-CMIT and triclosan) corresponding to the different derivatives strains. Cells were resuspended in fresh medium in the presence of imipenem and washed (when OD 600 was > 0Á5) in 20 mmol KH 2 PO 4 (pH 7Á0) buffer containing 1 mmol MgCl 2 and adjusted at 0Á375 OD 600 in the same buffer. In each microplate well, the bacterial suspension was fixed at 0Á18 OD 600 and nitrocefin (0Á2 lg ml À1 final concentration) was added to start the enzymatic reaction. The microplate was immediately placed into an Infinite M200 pro Tecan microplate reader (Tecan), and OD were monitoring at 490 nm for 1 h [START_REF] Matsumoto | Evaluation of multidrug efflux pump inhibitors by a new method using microfluidic channels[END_REF] to follow nitrocefin hydrolysis.

Preparation of membrane fractions, immunoblotting

Total protein fractions were prepared from bacterial exponential culture (1 OD 600 ), in the presence of respective concentration of preservatives for insusceptible strains. Cell pellets were solubilized at 96°C for porins analysis and 37°C for efflux pumps analysis. Bacterial membrane fractions were prepared from exponential cultures as previously described [START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF]. SDS-PAGE and immunoblot methods were carried out as previously reported using polyclonal antibodies directed against OmpA, denatured porins, AcrA, AcrB and TolC efflux pump components [START_REF] Simonet | Crucial domains are conserved in Enterobacteriaceae porins[END_REF][START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF]. Immunoblots were stained by colorimetric detection after incubation with alkalinephosphatase-conjugated secondary antibodies [START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF].

Extraction of flagella and immunodetection

Bacteria were recovered for LB agar plates, containing respective concentrations of preservatives for insusceptible strains, and resuspended at 1 OD 600 in phosphate buffer (0Á1 mmol, pH 7Á4). Cell suspension was pushed through syringe needles (0Á8*40 nm) 20 times to shear the flagellar propellers (Lef evre et al. 2010). Sheared cells were centrifuged at 14 000 g for 10 min at 4°C. Supernatant was precipitated with a final concentration of 15% trichloracetic acid (TCA), pelleted and washed twice in acetone 90% by centrifugation (14 000 g, 10 min, 4°C). In the next step, these pellets were resuspended in SDS-PAGE loading buffer and heated for 10 min at 96°C [START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF]. Antibodies anti-H7 flagellin (Difco, Detroit, Mich) were used at 1 : 20 000 fold dilution for immunoblot analysis [START_REF] Pradel | Contribution of the twin arginine translocation system to the virulence of enterohemorrhagic Escherichia coli O157:H7[END_REF]. Immunoblots were stained by chemiluminescence detection after incubation with horseradish peroxidase (HRP)conjugated secondary antibodies.

Mass spectrometry

The bands of interest were excised from the SDS-PAGE gel after staining and used for identification [START_REF] Zhang | Complex spatial organization and flagellin composition of flagellar propeller from marine magnetotactic ovoid strain MO-1[END_REF]. Tryptic digestion experiments of 1D bands were performed by a robotic sample preparation workstation (Freedom EVO 100, TECAN M€ annedorf, Switzerland) following multiple steps: washes, reduction and alkylation, digestion by trypsin (Sigma, St. Louis, MO, USA proteomics grade with 0Á025% ProteasMax) and extraction of tryptic peptides.

Tryptic peptides were separated and analysed using a positive ion mode and two scan events with a LTQ Velos Orbitrap mass spectrometer (Thermo Fisher Walthan, MA, USA) equipped with a nanospray ion source and coupled to a nano UPLC Ultimate NCS3500 (Dionex Sunnyvale, CA, USA). Spectra were processed by Proteome Discoverer software (Thermo Fisher Scientific), and the identification protein search was performed by MAS-COT (in situ licence) with Database : NCBInr 2012 (17 333 135 sequences), Taxonomy : Bacteria (Eubacteria) (10 365 170 sequences). Proteins were considered as identified by 2 unique peptides with ion significance threshold P < 0Á05 (>44 individual MASCOT ion score) or 5 consecutive b and y ions.

Real-time efflux

The efflux mechanism in bacteria has been implicated in exhibiting a significant alteration of susceptibility for antibiotics and biocides [START_REF] Nikaido | Broad-specificity efflux pumps and their role in multidrug resistance of Gramnegative bacteria[END_REF][START_REF] Ortega Morente | Biocide tolerance in bacteria[END_REF]. In Gram-negative bacteria, several efflux pumps have been identified in biocide-resistant strains [START_REF] Ortega Morente | Biocide tolerance in bacteria[END_REF]. To determine and compare the activity of efflux pumps present in the various resistant derivatives and the parental strain, a method previously described by [START_REF] Lieutaud | Inhibitors of antibiotic efflux by AcrAB-TolC in Enterobacter aerogenes[END_REF] was employed. This method makes it possible to follow the expel rate of a dye previously accumulated in the bacterial cell when an energy source (glucose) for the active efflux is added to the suspension [START_REF] Brunel | Polyamino geranic derivatives as new chemosensitizers to combat antibiotic resistant Gramnegative bacteria[END_REF][START_REF] Lieutaud | Inhibitors of antibiotic efflux by AcrAB-TolC in Enterobacter aerogenes[END_REF]. Briefly, for each strain, the different derivative-resistant strains inoculated with 20 ml of MHB were grown for 18 h at 37°C in the presence of respective preservative concentrations. All the subsequent steps were performed at room temperature. The cells were recovered by centrifugation and washed once in 20 mmol KH 2 PO 4 buffer (pH 7Á0) containing 1 mmol MgCl 2 (PPB). After a second centrifugation, the cell pellet was resuspended and adjusted to 0Á25 OD 600 in PPB. 1,20-Dinaphthylamine (1,2 0 DNA) (TCI-Europe SA, Zwijndrecht, Belgium) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma) were added at 32 and 5 lmol, respectively. The incubation was then performed overnight at 37°C without shaking. Aliquots were taken from the cell suspension, and bacteria were recovered by centrifugation (4000 g, 20 min) and resuspended in the same volume of PPB. The aliquot was then loaded (100 ll per well) into 96-well Greiner microplate (Greiner, Courtaboeuf, France), which was immediately placed into an Infinite M200 pro Tecan microplate reader. In the conditions used, the highest signal was obtained using an excitation wavelength OD 370 nm and an emission OD 420 nm . All the recordings were performed at a gain setting of 65. The fluorescence of cell suspension was observed for 150 s, and the efflux of 1,2 DNA was then triggered by rapid energization of cells by adding glucose (50 mmol final concentration), monitoring the fluorescence for another 450 s.

Results

Susceptibility of preservatives adapted strains to disinfectants

Table 2 presents the different MIC and MBC results against the disinfectants that are generally used in the cosmetic industry. Peroxyacid, ADD/PHMC and laurylamine dipropylenediamine used in the disinfection step were assayed. Susceptibilities were determined on the parental strain Ent. gergoviae ATCC 33028, their respective MIT-CMIT-and triclosan-resistant derivatives. The industrial isolates, for example 23-26, have been previously tested (P eriam e et al. 2014). MICs were tested in MH medium and MBC in Eugon LT medium. D1, D2 or D4 corresponds to 1, 2 or 4 days incubation, respectively. The use of D1, D2 and D4 allowed us to check the possibility of a delayed restart due to the presence of persister cells. Successive enumerations were performed according to ISO standards. No significant MIC differences were observed with MIT-CMIT-and triclosan-adapted strains compared to the parental strain (Table 2). For laurylamine dipropylenediamine present in Eugon LT, MBC was increased 8-16 times for MIT-CMIT-and triclosanresistant derivatives compared to the parental strain. No significant MBC differences were measured for the 3 days.

Susceptibility to antibiotics, preservatives and chemicals (EDTA, DOC)

The MICs for polymyxin B, nalidixic acid and b-lactams (imipenem, cefepime, ceftazidime) did not differ substantially between the resistant derivatives and their parental strain (Table 3). In the same way, no MIC variations were observed for triclosan and MIT-CMIT between ATCC, derivatives and cosmetic strains (no more than one dilution difference which is considered as no significant). Addition of PABN caused an increase in antibiotics tested susceptibility by no more than one dilution factor as for nalidixic acid of ATCC 33028 (parental strain) and resistant strains (M1-M4, T1-T3), indicating with the criteria of the 3 significative dilution factors that no PAbNsusceptible efflux could be detected. Only the polymyxin B susceptibility was increased in the presence of PAßN for all strains suggesting a similar synergistic effect on the Ent. gergoviae susceptibility (eightfold, respectively). This PAßN concentration (20 mg l À1 ) has been previously used to restore antibiotic susceptibility in Ent. aerogenesand Klebsiella pneumoniae-resistant strains [START_REF] Mamelli | New antibiotic molecules: bypassing the membrane barrier of gram negative bacteria increases the activity of peptide deformylase inhibitors[END_REF][START_REF] Pag Es | Efflux pump, the masked side of ß-lactam resistance in Klebsiella pneumoniae clinical isolates[END_REF][START_REF] Kuete | Antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype[END_REF]. In same assays, no effect was obtained for ß-lactam in the presence of PAßN and a subinhibitory concentration of cloxacillin (data not shown). Interestingly, the susceptibility to EDTA reached 80 mmol for MIT-CMIT-and triclosan-resistant derivatives strains that corresponded to about 16-fold increase compared to the parental strain. This susceptibility decrease could be associated with an adaptive resistance to EDTA-chelating action and outer membrane destabilizing strategy. The destabilization of the LPS layer induced by removal of divalent cations by EDTA could be reduced in adaptive strains, resulting in a preserved integrity of the outer membrane. No modifications in the susceptibility were observed with polymyxin B, a permeabilizing agent in the absence or in the presence of EDTA. In addition, a decrease of DOC susceptibility was also observed in the resistant strains. Interestingly, various papers reported a relationship between LPS modification and the susceptibility of Gram-negative bacteria to EDTA and detergents [START_REF] Bell | Pseudomonas aeruginosa outer membrane protein OprH: expression from the cloned gene and function in EDTA and gentamicin resistance[END_REF][START_REF] Liu | Overexpression of mltA in Edwardsiella tarda reduces resistance to antibiotics and enhances lethality in zebra fish[END_REF]). Together, these data suggest the involvement of outer membrane and particularly LPS modifications in bacteria biocides adaptation as precedently observed [START_REF] Chapman | Preservative tolerance and resistance[END_REF].

Impact of preservatives and disinfectants on the permeability of the outer membrane

Many biocide molecules exhibit a membranotropic effect inducing an alteration of membrane permeability [START_REF] Ortega Morente | Biocide tolerance in bacteria[END_REF]. Hence, we were inclined in knowing if a modification of the outer membrane barrier is liable for the emergence of resistant strains after biocide exposure. The assays using the determination of ß-lactamase release have been described as powerful tools to follow the possible change of membrane physiology [START_REF] Fadli | Thymus maroccanus essential oil, a membranotropic compound active on Gramnegative bacteria and resistant isolates[END_REF]. In this assay, nitrocefin is hydrolysed by the periplasmic ß-lactamase of Gram-negative bacteria, and the detection of chromogenic product is an indicator of the outer membrane (OM) permeability [START_REF] Jones | In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other betalactam compounds[END_REF].

We first tested the OM permeabilizing potential of five preservatives (methylisothiazolinone, MIT-CMIT, pentylene glycol, levulinic acid, triclosan) and one disinfectant (peroxyacid) on the parental ATCC strain. Only the peroxyacid showed an increase of nitrocefin hydrolysis due to a permeabilizing effect obtained with concentrations between 0Á20% and 0Á5%, which corresponds to the concentration range of use in cosmetic industries (Fig. 1).

Regarding the resistant strains, the amount of hydrolysed nitrocefin was not significantly changed in MIT-CMIT (M1-M4)-and in triclosan (T1-T3)-resistant isolates

0Á03 0Á007 0Á015 0Á06 0Á06 M1 0Á015 0Á06 0Á007 0Á015 0Á06 0Á125 M2 0Á015 0Á06 0Á015 0Á015 0Á06 0Á5 M3 0Á015 0Á06 0Á015 0Á015 0Á06 1 M4 0Á015 0Á03 0Á015 0Á015 0Á06 1 T1 0Á015 0Á03 0Á015 0Á015 0Á06 0Á5 T2 0Á015 0Á015 0Á007 0Á015 0Á06 0Á5 T3 0Á015 0Á03 0Á007 0Á015 0Á06 0Á5
(data not shown). This suggests that no important alteration of the outer membrane permeability promoting the release of hydrolysed nitrocefin could be present in the resistant isolates.

Outer and inner membrane proteins in the various strains

Analysis of total bacterial proteins by SDS-PAGE showed no difference in the migration of molecules in the size range of 30-45 kDa molecular weights. Similarly, the immunodetections of OmpA, porins, and components of efflux pump AcrA, AcrB and TolC reported no variation in the signal intensity or in the migration of these key membrane proteins (Fig. 2). Interestingly, after SDS-PAGE evaluation of the bacterial compartments, various differences were observed in the profile: in the OM fraction of the resistant strain, (i) we observed an additional band migrating around 35-kDa mark, (ii) a more intense upper band at about 40 kDa and (iii) the appearance of a slightly intermediate band between them. The three instances have been pointed as c, d, e in Fig. 3. Various differences were also observed on the profiles of Ent. gergoviae strains, eg23 and eg26, isolated from contaminated cosmetics, for which we have previously reported an insusceptibility for various preservatives (P eriam e et al. 2014). They exhibited three bands instead of the two detected in the parental strain ATCC 33028. The 35-to 45-kDa part of the SDS-PAGE exhibited some similarity with the profile of the MIT-CMIT-resistant strains.

Flagellin and peroxiredoxin identification in various strains

Mass spectrometry analyses were carried out to identify the proteins detected in the SDS-PAGE patterns of resistant strains. The respective six bands, a to f (Fig. 3), were excised from the gels, and the proteins were retrieved and used for mass spectrometric determinations. The analysis identified several proteins-the lower band as OmpA-the upper band one for the parental strain, and two bands for the derivative resistant strains were identified as flagellin protein (Table 4). A higher molecular weight flagellin was detected in the resistant strain M1. Immunodetection using anti-H7 flagellin matched with two bands: one located in the OM fraction, close to 40 kDa for M1, and one identified in the supernatant fraction, close to 38 kDa. The immunoreactive protein was observed in the M1-resistant strain, the triclosan-resistant derivatives (T1-T3) and strains isolated from cosmetic products (eg23 and eg26) (Fig. 4). Furthermore, a 25-kDa product was observed in the cytosolic fraction of the M1 strain. Six peptides showing 46% sequence coverage (MASCOT search on NCBInr Database reduced to Bacteria (15 365 170 entries) were obtained (Table 4). The mass spectrometry analyses fitted in with an antioxidant enzyme, peroxiredoxin that controls cytokine-induced peroxide levels, for this 25-kDa protein.

Real-time efflux

Bacteria are able to expel a large variety of compounds such as antibiotics, biocides, detergents or dyes (Nikaido and Pag es 2012). 1,2 DNA, a fluorescent dye, is a substrate of the AcrAB efflux pump belonging to RND family, and it has been used to monitor the bacterial efflux.

A decrease in the dye's fluorescence intensity allows the assessment of the activity of active drug pump [START_REF] Brunel | Polyamino geranic derivatives as new chemosensitizers to combat antibiotic resistant Gramnegative bacteria[END_REF][START_REF] Lieutaud | Inhibitors of antibiotic efflux by AcrAB-TolC in Enterobacter aerogenes[END_REF]. When the assays were carried out, the resistant strains showed a significant variation in the efflux activity compared to those obtained with the parental strain (Fig. 5). An efflux activity corresponding to about 75-60% of the level determined in the parental strain was observed for MIT-CMIT-resistant strains (M1-M4) that produced very similar curves. Interestingly, a stronger reduction was observed with the triclosan-resistant strains (T1-T3) with only 35-18% of the efflux measured compared to the parental strain (Fig. 5). These results suggest that a reduction of the dye efflux efficacy was reached in the resistant strains.

Discussion

The objective of this work was to understand the Ent. gergoviae adaptive mechanisms to preservatives used in the case of recurrent contaminations. To this aim, we obtained and characterized resistant isolates which are able to grow in the presence of increased concentrations of preservatives. Different profiles were observed depending on the preservatives used during the selection as illustrated by the respective MIT-CMIT and triclosan MICs and the laurylamine MBC measurements.

Importantly, the resistant strains selected with increased concentrations of MIT-CMIT or triclosan presented no variation in antibiotic susceptibility in Mueller-Hinton broth containing twofold serial dilutions of each antibiotic. This indicated that no cross-adaptive mechanisms between these two preservatives and antibiotics were selected or expressed such as the activation of a common efflux system or membrane impermeability (AcrAB overproduction, loss of porins). Various studies concerning biocide adaptation, with a special attention to triclosan, have produced contradictory results about the relationship between biocides and antibiotics and the involvement of general mechanisms such as the efflux [START_REF] Chuanchuen | Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ[END_REF][START_REF] Condell | Efficacy of biocides used in the modern food industry to control Salmonella enterica, and links between biocide tolerance and resistance to clinically relevant antimicrobial compounds[END_REF][START_REF] Sheridan | The potential for biocide tolerance in Escherichia coli and its impact on the response to food processing stresses[END_REF][START_REF] Rensch | Analysis of triclosan-selected Salmonella enterica mutants of eight serovars revealed increased aminoglycoside susceptibility and reduced growth rates[END_REF]. Moreover, regarding nalidixic acid, MIT-CMIT and triclosan, PAßN used at a concentration previously reported to block efflux activity in Ent. aerogenes-, Kl. pneumoniae-and Escherichia coliresistant strains [START_REF] Mamelli | New antibiotic molecules: bypassing the membrane barrier of gram negative bacteria increases the activity of peptide deformylase inhibitors[END_REF] AcrAB-TolC efflux pump as previously reported [START_REF] Condell | Efficacy of biocides used in the modern food industry to control Salmonella enterica, and links between biocide tolerance and resistance to clinically relevant antimicrobial compounds[END_REF][START_REF] Rensch | Analysis of triclosan-selected Salmonella enterica mutants of eight serovars revealed increased aminoglycoside susceptibility and reduced growth rates[END_REF][START_REF] Rushton | Key role for efflux in the preservative susceptibility and adaptive resistance of Burkholderia cepacia complex bacteria[END_REF].

Interestingly, no alteration in porins expression and in ß-lactams susceptibility was observed, indicating that a change in the porin-associated permeability is not involved in the various resistant strains, in contrast to previous reports [START_REF] Chapman | Preservative tolerance and resistance[END_REF][START_REF] Ortega Morente | Biocide tolerance in bacteria[END_REF]. Concerning outer membrane proteins and bacterial adaptation to isothiazolinones, different results have been reported in Pseudomonas aeruginosa. It was described that the Ps. aeruginosa adaptation to MIT and CMIT was associated with the decrease of a 35-kDa outer membrane protein named T-OMP and with a decreased susceptibility towards the individual components of MIT-CMIT (Br€ ozel and Cloete 1994). Moreover, T-OMP production was observed de novo after removal of the isothiazolone biocides, while the treated cultures still exhibited some residual tolerance towards these compounds. The authors suggested that T-OMP expression was linked to isothiazolone; however, T-OMP itself was not the cause of the decreased susceptibility [START_REF] Winder | Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1[END_REF]. It was concluded that the T-OMP expression may be specific for thiol-interactive biocides in Ps. aeruginosa. No additional studies have been carried out to determine the biological function of T-OMP and its possible likeness with OMPs in Enterobacteriaceae.

These results suggest that the exposure of Ent. gergoviae to MIT-CMIT or triclosan under the conditions used in this study did not activate a global control cascade as previously reported in the case of Ent. aerogenes and imipenem exposure that triggers a Mar operon response [START_REF] Bolla | Membrane permeability and regulation of drug "influx and efflux" in enterobacterial pathogens[END_REF]. This is interesting to mention that a stress response involving stress regulons has been previously reported for E. coli and Ps. aeruginosa during preservative exposure [START_REF] Abdel-Malek | Antimicrobial susceptibility changes and T-OMP shifts in pyrithionepassaged planktonic cultures of Pseudomonas aeruginosa PAO1[END_REF][START_REF] Yu | Signature gene expression profile of triclosan-resistant Escherichia coli[END_REF][START_REF] Zhou | Involvement of outer membrane proteins and peroxidesensor genes in Burkholderia cepacia resistance to isothiazolone[END_REF].

In contrast, MIT-CMIT-and triclosan-resistant derivatives were less susceptible to laurylamine dipropylenediamine compared to the parental strain. This indicates that the mechanisms selected in the derivative strains can provide a cross-insusceptibility towards some preservatives and disinfectants. This bacterial adaptation may impair the activity of various disinfectants used in practice to address Ent. gergoviae contaminations. The MIT-CMIT is an isothiazolinone biocide used by the cosmetic company for the disinfection of cosmetic production and distribution systems, destructing protein-thiols and producing free radicals [START_REF] Williams | The mechanism of action of isothiazolone biocide[END_REF]. MIT and CMIT interact via redox reaction with thiols to form disulphites, cystine and biocide dimers (thio-acyl chloride) highly reactive in the case of CMIT (Collier et al. 1990a(Collier et al. , b, 1991)). The thio-acyl chloride is associated with the antimicrobial activity of isothiazolinones by reacting with glutathione or amines (Collier et al. 1990b). The ADD/ PHMC combination and the laurylamine dipropylenediamine have surfactant properties. Biguanides seem to cause a strong destabilization of the membrane causing leakage of cytoplasmic contents [START_REF] Broxton | Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli[END_REF][START_REF] Ikeda | Interaction of a polymeric biguanide biocide with phospholipid membranes[END_REF][START_REF] Allen | The response of Escherichia coli to exposure to the biocide polyhexamethylene biguanide[END_REF]. Peroxygen molecules have strong oxidative property that attack essential cell components and cell membranes and additionally produced active hydroxyl radicals. However, peroxyacid effects seemed less effective on the MIT-CMIT-or triclosan-resistant strains compared to the parental strain, suggesting that membrane changes provide some protection towards peroxygens molecules in resistant derivatives.

Analyses of membrane proteins potentially involved in the permeability adaptation such as efflux pumps or porins showed no significant differences between derivative resistant strains and the parental strain. The main difference observed in the M1 adapted strain and strains isolated from cosmetics corresponds to a significant expression of a flagellin protein identified by mass spectrometry. Overexpression of flagellin components has been previously reported in E. coli triclosan-resistant mutants [START_REF] Bailey | Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a speciesspecific response, including drug detoxification[END_REF][START_REF] Sheridan | Proteomic and phenotypic analysis of triclosan tolerant verocytotoxigenic Escherichia coli O157:H19[END_REF]. Interestingly, in these strains that exhibit a modification of flagellin expression, we also observed a decrease in EDTA and DOC susceptibility, indicating involvement of outer membrane as a dynamic and permeability barrier face to preservatives as mentioned for Ps. aeruginosa [START_REF] Zhou | Involvement of outer membrane proteins and peroxidesensor genes in Burkholderia cepacia resistance to isothiazolone[END_REF]. In a recent publication, Kirkpatrick and Viollier reported the intricate relationship between efflux system and flagella functional assembly [START_REF] Kirkpatrick | Synthetic interaction between the TipN polarity factor and an AcrAB-family efflux pump implicates cell polarity in bacterial drug resistance[END_REF]. This original mechanism involved in drug insusceptibility is associated with the control of flagellin expression in Caulobacter crescentus. This study presents some similarities with the results associated with Ent. gergoviae triclosan-resistant mutants, for example expression of flagellin, decrease of dye efflux and envelope modification. We hypothesized that this variation could be associated with modifications of bacterial surface or in the modification of the energy level of bacterial membrane [START_REF] Thiolas | Successive emergence of Enterobacter aerogenes strains resistant to imipenem and colistin in a patient[END_REF][START_REF] Kastbjerg | Triclosan induced aminoglycoside-tolerant Listeria monocytogenes isolates can appear as small-colony variants[END_REF]. Interestingly, the triclosan-resistant strains exhibit a noticeable difference in the dye efflux that requires a membrane energy source as previously demonstrated [START_REF] Lieutaud | Inhibitors of antibiotic efflux by AcrAB-TolC in Enterobacter aerogenes[END_REF]). In the MIT-CMIT-resistant strain M1, an overproduction of peroxiredoxin has been observed. Peroxiredoxins are cysteine-based peroxidases that act as redox sensors and biomarkers of oxidative stress. They are detoxifying enzymes expressed in a wide range of eubacteria for reactive species as hydroperoxides [START_REF] Wood | Structure, mechanism and regulation of peroxiredoxins[END_REF][START_REF] Hall | Typical 2-Cys peroxiredoxinsstructures, mechanisms and functions[END_REF]. The cysteines are oxidized to reduce peroxides by forming an intermolecular disulphide bond, and the disulphide-bonded peroxiredoxins are subsequently reduced and reactivated by thiol-containing reductants such as alkyl hydroperoxidase subunit F (AhpF) and thioredoxin (TrxA). Interestingly, the isothiazolones are biocides which have a growth inhibition activity associated with a thiol-interactivity (Collier et al. 1990a(Collier et al. ,b, 1991)). Moreover, thiol-containing compounds such as cysteine, which is a thiol amino acid, are able to neutralize activity of CMIT (Collier et al. 1990a). Consequently, there is an evident relationship between isothiazolinones activity and overexpression of peroxiredoxin. AhpC (alkyl hydroperoxidase subunit C), one of the best characterized 2-cysteine peroxiredoxins, utilizes AhpF as a reductant to compose an NAD(P)H-dependent peroxidase system. Expression of AhpC is activated by OxyR, which is a central regulator of the oxidative stress response in a number of bacteria [START_REF] Hall | Typical 2-Cys peroxiredoxinsstructures, mechanisms and functions[END_REF][START_REF] Zhou | Involvement of outer membrane proteins and peroxidesensor genes in Burkholderia cepacia resistance to isothiazolone[END_REF]). Interestingly, it has been recently demonstrated that Burkholderia cepacia strains, resistant to isothiazolinones, overexpressed the transcriptional activator OxyR [START_REF] Zhou | Involvement of outer membrane proteins and peroxidesensor genes in Burkholderia cepacia resistance to isothiazolone[END_REF].

The present results established an additional relationship between the isothiazolinones biocides, the cellular interactions response including bacterial oxidative stress answer and detoxifying enzymes regulation and the bacterial tolerance to biocides.

To conclude, it is clear that the resistant strains obtained in this study exhibited no cross-adaptive mechanism altering the antibiotic activity; in contrast, a crossinsusceptibility towards preservatives and disinfectants was observed. It is important to mention that no increase of efflux activity or modification of porins level was detected in the various MIT-CMIT-and triclosan-resistant strains. Interestingly, several insusceptible strains and the two strains issued from contaminated cosmetics exhibited a strong modification of bacterial envelope with an overproduction of flagellin, but the porins and OmpA expression was uniform. This suggests that the same adaptive mechanisms may be present in the laboratory-induced insusceptible strains and the industrial isolates.

  polymyxin B, EDTA was tested at 1 mmol. *For DOC, complete solubilization was not possible beyond the concentration >16 lg ml À1 .

Figure 2 Figure 1

 21 Figure2Immunodetection of major outer membrane proteins and components of efflux pump AcrAB-TolC, Total bacterial proteins were separated by SDS-PAGE, electrotransferred onto nitrocellulose and analysed by immunoblotting as previously described[START_REF] Simonet | Crucial domains are conserved in Enterobacteriaceae porins[END_REF][START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF]. Antibodies directed against OmpA, OmpC, OmpF, AcrA AcrB, and TolC were used. Immunoblots were stained by chemiluminescence detection after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Only the relevant part of the sheet was presented.

Figure 3

 3 Figure3SDS-PAGE profiles for total membrane proteins, outer membrane proteins and soluble proteins. Bacterial membrane fractions were prepared from exponential cultures as previously described[START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF]. SDS-PAGE were carried out and stained as previously reported[START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF]. The total membrane proteins, outer membrane proteins and cytosolic proteins (cytoplasmic + periplasmic proteins) were indicated. d corresponds to the standard molecular weight; a, c, d = flagellin; b, e = outer membrane protein A (OmpA); f = peroxiredoxin.

Figure 4 Figure 5

 45 Figure4Immunodetection of flagellin. ATCC strain, eg23 isolate and resistant derivates MIT-CMIT (M1 to M4) were cultivated. Bacterial fractions were prepared from exponential cultures and SDS-PAGE were carried out and stained as described in Materials and Methods. The immunodetection was carried out with polyclonal antibody against H7 flagellin of Escherichia coli. Immunoblots were stained by chemiluminescence detection after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Only the relevant part of the sheet was presented.

Table 1

 1 Bacterial Enterobacter gergoviae strains and corresponding derivative-resistant strains used in this study

	Origin	Name in this study
	Cosmetic Industry (P eriam e et al. 2014)	eg23
	Cosmetic Industry (P eriam e et al. 2014)	eg25
	Cosmetic Industry (P eriam e et al. 2014)	eg26
	Ent. gergoviae, ATCC 33028
	selected with MIT-CMIT
	(at 1Á875Á10

À4

%, 3Á75Á10 À4 %, 4Á5Á10 À4 % and 6Á10 À4 %, respectively) M1, M2, M3, M4

Ent. gergoviae, ATCC 33028 selected with triclosan (at 5, 10 and 20 lg ml À1 , respectively)

T1, T2, T3

Table 2

 2 MICs and MBCs of disinfectants for the parental strain and MIT-CMIT (M1-M4) or triclosan (T1-T3)-resistant derivatives

						Laurylamine dipropylamine
		ADD/PHMC (%)		Peroxyacid (%)		(%)
		MIC	MBC	MIC	MBC	MIC	MBC
	Time	D1	D1, D2, D4	D1	D1, D2, D4	D1	D1, D2, D4
	ATCC 33028	0Á015

Table 3

 3 Susceptibilities of the Enterobacter gergoviae ATCC 33028 strain and its derivative-resistant strains to EDTA, DOC (deoxycholate), MIT-CMIT, triclosan and different antibiotics (poly-

		CAZ	(lg ml À1
	myxin, nalidixic acid, imipenem, ceftazidime, cefepime) with or without efflux pump inhibitor (PAbN)	DOC EDTA POL POL+ POL+ Nal Nal+ IMI IMI+	strains (lg ml À1 ) (mmol) (lg ml À1 ) EDTA PAbN (lg ml À1 ) PAbN (lg ml À1 ) PAbN

Table 4

 4 Identification of detected proteins in the resistant strains presented in SDS-PAGE outer membrane proteins

	Sample	Gi	Mascot Score	Mass (Da)	Matches	Sequences	Name	Coverage (%)
	a	gi|56383043	555	36 326	15	7	Flagellin [Escherichia coli]	2 7
	b	gi|376387855	666	38 239	107	11	Outer membrane protein A	22
							[Klebsiella oxytoca 10-5243]
	c	gi|56383043	2156	36 326	72	7	Flagellin [E. coli]	2 7
	d	gi|56383043	1147	36 326	36	4	Flagellin [E. coli]	2 0
	e	gi|317491503	277	37 996	102	7	OmpA transmembrane domain-	24
							containing protein
							[Enterobacteriaceae bacterium
							9-2-54FAA]
	f	gi|345298292	536	20 792	21	19	Peroxiredoxin [Enterobacter
							asburiae LF7a]

  [START_REF] Kuete | Antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype[END_REF] did not increase the respective drug activity whatever the strain tested. In addition, no significant change was noted for ß-lactam molecules, which have been reported to be substrates for Kl. pneumonia AcrAB efflux pump (Pag es et al. 2009). Moreover, immunodetections of the major pump AcrAB-TolC reported no significant variation in the expression level of efflux components and analysis of ß-lactamase activity indicated no change in the outer membrane permeability of resistant mutants. Consequently, in the resistant derivatives, the preservative insusceptibility may use other mechanisms than those involved in antibiotic resistance such as

	ATCC	ATCC3	Ladder	Ladder	M1	M1	M2	M3	M4	eg23
	33028	3028	broad	Low	(OM)
	(OM)			range
	32 kDa
	eg26	eg26	Ladder	Ladder	ATCC	T1	T2	T3	eg23	eg23
	(OM)		Low	broad	33028					(OM)
			range
	32 kDa

Acknowledgements

The authors thank R. Lebrun, S. Lignon, E. Baudelet and S. Audebert for performing identification analyses by LC/ MSMS mass spectrometry. The authors thank L. Fei Team for provide us anti-H7 flagellin, V. Lome for his helpful advices regarding real-time efflux experiments, N. Philippe for her stimulating discussion on bacterial envelope. We greatly appreciate the S. Dam s' help for his careful reading of the manuscript. This work was supported by Aix-Marseille Universit e. M.P. is recipient from a PACA-Entreprise thesis fellowship.

Conflict of Interest

No conflict of interest declared.


Zachary Pezzementi

Sandrine Voros

Gregory D Hager

Articulated Object Tracking By Rendering Consistent Appearance Parts

We describe a general methodology for tracking 3-dimensional objects in monocular and stereo video that makes use of GPU-accelerated filtering and rendering in combination with machine learning techniques. The method operates on targets consisting of kinematic chains with known geometry. The tracked target is divided into one or more areas of consistent appearance. The appearance of each area is represented by a classifier trained to assign a class-conditional probability to image feature vectors. A search is then performed on the configuration space of the target to find the maximum likelihood configuration. In the search, candidate hypotheses are evaluated by rendering a 3D model of the target object and measuring its consistency with the class probability map. The method is demonstrated for tool tracking on videos from two surgical domains, as well as in a human hand-tracking task.

I. INTRODUCTION

There are many applications in which the fundamental problem is to track an articulated object with changing appearance in a changing background. Examples range from tracking rigid objects in motion [START_REF] Harris | Tracking with Rigid Objects[END_REF] to tracking robot arms or hands [START_REF] Ruf | Visual tracking of an end-effector by adaptive kinematic prediction[END_REF], [START_REF] De La Riviere | Image-based analysis for modelbased tracking[END_REF] to tracking one or more humans in images [START_REF] Gavrila | The visual analysis of human movement: a survey[END_REF]. In almost all cases, it is reasonable to assume that a reasonably accurate kinematic model is available for the target in question. However, what is not known, a priori, is the appearance of the target and, more importantly, what distinguishes the target most effectively from the background.

The problem of simultaneously learning and tracking a model has seen a great deal of activity in recent years. Early work developed per-pixel generative models for the foreground only [START_REF] Mckenna | Tracking colour objects using adaptive mixture models[END_REF] (resp. background only [START_REF] Stauffer | Adaptive background mixture models for real-time tracking[END_REF]). Background (resp. foreground) objects were then detected as "outliers" to the process. More recent work such as [START_REF] Yu | Monocular video foreground/background segmentation by tracking spatial-color gaussian mixture models[END_REF] develops both foreground and background appearance models using spatially augmented GMMS. In work specific to tracking, [START_REF] Avidan | Ensemble tracking[END_REF] and [START_REF] Grabner | On-line boosting and vision[END_REF] develop and maintain an ensemble of weak classifiers over time by re-weighting, adding or rejecting classifiers. Lu [START_REF] Lu | A nonparametric treatment for location/segmentation based visual tracking[END_REF] maintains a "bags of image patches" appearance model using a temporal-adaptive importance resampling procedure. In general, in these approaches a classifier is trained over a set of images using selected features (for instance color histograms in [START_REF] Zhang | Robust visual tracking via pixel classification and integration[END_REF] or a combination of local orientation histogram and pixel colors in [START_REF] Avidan | Ensemble tracking[END_REF]), to identify and discriminate foreground from background.

In the work cited above, the tracked target has no a priori geometric structure, and tracking amounts to maintaining some notion of location or rough target outline over time. Often, there is a need for information beyond location. Early Laboratory for Computational Science and Robotics (LCSR), Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218 {zap, svoros, hager}@cs.jhu.edu work in human tracking made use of local features such as edges [START_REF] Harris | Tracking with Rigid Objects[END_REF], [START_REF] Gavrila | The visual analysis of human movement: a survey[END_REF] to optimize over body pose, building upon several systems for tracking rigid or low-DOF articulated 3D objects which rely upon visual contours [START_REF] Drummond | Real-time visual tracking of complex structures[END_REF], [START_REF] Rosenhahn | Pose Estimation of 3D Free-Form Contours[END_REF], [START_REF] Lowe | Fitting parameterized 3-d models to images[END_REF], [START_REF] Rehg | Visual tracking of high dof articulated structures: An application to human hand tracking[END_REF]. Although this may work for models with well-defined edges before uncluttered backgrounds, these contours can not be reliably detected in many real-world settings. For instance, the surgical setting from which most of our examples are drawn is rife with features which are detected as spurious edges by all standard edge detectors, as seen in Figure 1, and which would confuse any solely edge-based approach. Dealing with so many outliers would likely be very expensive. These same challenging visual effects, specularities and inconsistent lighting, occlusion, and small depth of field, pose problems for other local features as well. We opt, instead, to take a more "image-based" approach, making use of all visible parts of the target object, rather than only those which are most distinctive.

Also in the human tracking field, other local features such as optical flow [START_REF] Li | 3-d motion estimation in model-based facial image coding[END_REF], template-matching [START_REF] Cascia | Fast, reliable head tracking under varying illumination: An approach based on registration of texture-mapped 3d models[END_REF] or object texture [START_REF] Bregler | Learning and recognizing human dynamics in video sequences[END_REF] have been employed. Other work has explored interest point correspondences [START_REF] Grabner | Learning features for tracking[END_REF], [START_REF] Vacchetti | Stable real-time 3d tracking using online and offline information[END_REF]. The 3D pose recovery is then either formulated as an inverse kinematics problem or a constrained optimization problem, the latter formulation typically being more robust over time [START_REF] Gavrila | The visual analysis of human movement: a survey[END_REF]. Recent work on human tracking [START_REF] Sminchisescu | Bm3e: Discriminative density propagation for visual tracking[END_REF] uses conditional Bayesian mixtures of kernel-induced experts to build "bottom-up" discriminative models of pose probabilities from features taken from the silhouette of the target. In [START_REF] Ramanan | Tracking people by learning their appearance[END_REF], the authors detect a model of parts consisting of parallel edges, then learn appearance models from those detections to use for tracking within the sequence. However, in most of this work, the focus is on detection, and tracking is treated as a "sequential detection problem" rather than an optimization over the configuration space of the target.

Thus, despite this volume of work, it is unfortunately still the case that there are few general-purpose tools for robust and efficient tracking of kinematic structures with fully or approximately known geometry and unknown appearance. Our work aims to create such a tool by combining statistical learning methods to create a generative model of appearance similar to [START_REF] De La Riviere | Image-based analysis for modelbased tracking[END_REF], [START_REF] Lu | A nonparametric treatment for location/segmentation based visual tracking[END_REF], [START_REF] Speidel | Automatic image-based analysis for context-aware assistance in minmally invasive surgery[END_REF], [START_REF] Zhang | Robust visual tracking via pixel classification and integration[END_REF] with a structured, kinematically-based optimization to best align the geometric model with the data as [START_REF] Gavrila | 3-d model-based tracking of humans in action: a multi-view approach[END_REF] or [START_REF] Bregler | Learning and recognizing human dynamics in video sequences[END_REF]. Since the geometry of the model is known, one can view this problem as one of projecting the model into one or more images, then associating each projected part with a model of approximately uniform appearance, using image information. As we will show, our algorithms rely on relatively simple image filtering, 3D projection, and appearance evaluation algorithms. As a result, most calculations can be performed by commodity GPU hardware, making the achievement of real-time performance relatively straightforward.

II. TRACKING FROM RENDERED MAPS

Our tracking method consists of two components: 1) an appearance modeling step that computes a probability of class membership for each appearance class and for each pixel, and 2) an optimization step or filtering step that computes the configuration of the object from this probability map. Here, we present the underpinnings of the latter and defer our discussion of appearance classification to Section III-A.

As a starting point, we note that any serial chain mechanism consisting of rigid bodies can be represented by a minimal set of variables that in turn define the object's configuration space [START_REF] Lozano-Perez | An algorithm for planning collision-free paths among polyhedral obstacles[END_REF]. In what follows, let x denote some parameterization of the configuration of an object. We denote an image by I and, pixel-related feature vectors in the image by v ∈ I. Let C denote a parameterization of an appearance model on image feature vectors and let θ f = {C 1 , C 2 , . . . C m } represent the set of appearance models for a "foreground" object in question. We denote the background appearance class by B and define θ = θ f ∪ {B}. We assume that, for each appearance class in question, we can "render" the area of the target into the image to create a mask which in turn identifies a set of these feature vectors. We write C(x) (resp. B(x)) to denote the set of pixel vectors in the image corresponding to the mask generated for a given class and an object configuration. These form a strict partition on all feature vectors in an image. Finally, we distinguish between estimated quantities and random variables by writing them with a "hat" over them, i.e. x vs. x.

There are a variety of estimation-theoretic paradigms for posing and solving the problem of configuration estimation. Two of immediate interest are 1) maximum likelihood estimation and 2) time-series filters that rely on importance sampling. Both methods rely on the ability to evaluate the likelihood function at a particular object configuration. Assuming independence of feature vectors given a classification, the likelihood function can be written as

P (I | x, θ) = C∈{θ} v∈C(x) P (v | C) (1) Define L(v, C) = -log P (v | C).
Taking the negative log of (1), we have

-log P (I | x, θ) = C∈θ v∈C(x) L(v | C) (2)
= C∈θ f v∈C(x) L(v | C) + v∈I L(v | B) - C∈θ f v∈C(x) L(v | B) (3) =c + C∈θ f v∈C(x) (L(v | C) -L(v | B)) (4)
where c is a constant factor that varies from image to image, but is independent of x within a given image.

As a consequence, we can now write (1) in an equivalent form as

P (I | x, θ) = κ C∈θ f v∈C(x) P (v | C) P (v | B) (5)
It is interesting to note that, for a single foreground class, the resulting expression takes the form of a likelihood ratio. One way of performing tracking is to simply compute the maximum likelihood pose at each step, i.e. to solve

x = arg max x P (I | x, θ) (6)
= arg min

x C∈θ f v∈C(x) L(v | C) -L(v | B) (7)
In general, this optimization can be quite complex, as the search space can be high-dimensional and the objective function is not guaranteed to be convex. Our current methods for solving it are described in Section III. However, for special cases, the solution is immediate. For example, when computing image location (i.e. object translation when motion lies approximately in a plane) with a single foreground class we can consider the mask as a kernel. Then, ( 7) can be solved by finding the location of the global maximum when convolving the target mask over the difference between foreground and background classification log probabilities.

Local optimization can be easily accomplished using the mean shift algorithm [START_REF] Cheng | Mean shift, mode seeking, and clustering[END_REF]. Indeed, exactly this approach is applied in [START_REF] Lu | A nonparametric treatment for location/segmentation based visual tracking[END_REF]. Finally, we note that, given a method for sampling the likelihood function, methods for developing a particle filter for tracking configuration are well-established [START_REF] Doucet | Sequential Monte Carlo Methods in Practice[END_REF].

III. IMPLEMENTATION

The previous section provides a very general framework for the design of articulated tracking design from learned appearance models. We now turn to the specific choices made in our current implementation. We first describe our appearance modeling Section III-A, then discuss rendering and optimization details in Section III-B.

A. Appearance Learning

Our appearance model computes class-conditional probabilities from color and texture features extracted from the image, as described in the applications in Section IV. We currently assume at least one labeled image of the target is available. Using the labeled image, we apply linear discriminant analysis (LDA) to compute a linear dimensional reduction using the most discriminating features. Then, one of many possible statistical learning techniques is used to train a model capable of producing class probability estimates. In the example applications that follow in Section IV, both histograms and Gaussian mixture models are used, though the field of machine learning provides a wealth of other possibilities.

The initial model training is currently done by hand by aligning or hand-segmenting a model to one or more images of the data set, as shown in Figure 2. As we discuss in Section V, more automated training is possible, but we have not fully developed this aspect of the method yet.

In the results presented in this paper, we have found it sufficient to train models on one or two images in the sequence without further adaptation. Preliminary tests did not show a large variation in the performance of different classifiers on this task.

B. Rendering and Optimization Details

In our implementation, the image mask is evaluated by standard graphical rendering techniques given a 3dimensional model of the object: the object models were created in Pro/ENGINEER from measurements of the objects and exported as Wavefront triangular meshes. Articulated objects were modeled by part and then configured at rendertime. Rendering was performed using OpenGL to take advan- tage of hardware-acceleration. For stereo video sequences, this step required a known camera calibration to establish the relationship between the 3D model and its projection on the image plane. Camera(s) calibration(s) were obtained using Matlab/OpenCV Calibration Toolbox [START_REF] Strobl | Camera calibration toolbox for matlab[END_REF].

The results in this paper are all generated using maximum likelihood estimation. In order to optimize (1), we elected to use the Nelder-Mead downhill simplex method [START_REF] Nelder | A simplex method for function minimization[END_REF] since it requires the computation of only function values, not derivatives, and is robust to noisy minimization functions. Since we are projecting the entire tool into the image, local optimization from the previous target configuration typically converges reliably, provided there is sufficient overlap from the previous frame. Given the relatively slow motion, and the relatively high dimensionality of the problem, we found that spatial importance sampling did not provide a noticeable improvement over MLE.

In addition to the use of hardware-based rendering, we have performed preliminary tests of the time required for each step of the algorithm using a GPU implementation of GLSL shaders [START_REF] Kessenich | The OpenGL Shading Language v 1.20[END_REF]. Our current results are shown in Table I. The time required for applying whole-image filters of two sizes is given, as well as the number of such operations required for feature extraction. The timing of the generation of the probability map is also estimated, as well as the two parts of the image dot product that make up the evaluation of the objective function. The rows of Table II show the number of computations of the objective function expected to be possible at each frame-rate, based on those timings, with real-time performance expected in the final configuration, that of a high-end gaming system.

The hardware of the testing machine consisted of a dualcore 3-GHz Intel CPU, 2GB of RAM and an nVIDIA GeForce 8600GTS for graphics processing. Input images were 640-x-480 resolution. Runtime on each sequence was around 800ms for feature extraction and classification and 4 to 5 seconds for minimization in each frame. A realistic GPU implementation could make use of a higher-end graphics card such as a GeForce 8800GTX which has four times as many stream processors as the test system's card as well as additional video memory. The last two rows of Table II assume speed-up factors of 4 and 8 from substituting one or two of these cards respectively. Other optimizations, such as down-sampling of the input images or only classifying pixels in the neighborhood of the target, as well as handoptimization of the operations presented above or implementation in CUDA [START_REF] Corporation | NVIDIA CUDA Compute Unified Device Architecture Programming Guide v 1.1[END_REF], could yield significant further speedups. Real-time performance could therefore be achieved for relatively high-dimensional models using widely-available hardware.

In summary, the parameters that a user must set consist of the following: For appearance modeling, the number of appearance classes to use to represent the model must be chosen, as well as the number of components to use in the mixture model and the size of the feature extraction filters (which should be related to the size of input images). For optimization, assuming the use of downhill simplex, one must set the initial simplex and choose the termination conditions, i.e. convergeance criteria for absolute error or its derivative, or maximum iterations.

IV. RESULTS

We have applied the tracking method to a variety of monocular and stereo tracking problems. These include monocular tool tracking in microscopic sequences, tracking two articulated tools in stereoscopic video of robotic surgery, and monocular hand tracking. We also provide results from a simulated video data set. Each of the complete video sequences is available in the supplementary materials, as well as via web on the CIRL web-site.

On the simulated sequence, the ground truth configuration is known, and accuracy is measured by the magnitude of difference in the configuration estimate from the true configuration, ||x-x|| ||x|| . Since the true configuration is not available for the real sequences, accuracy is instead estimated by the pixel overlap of the rendered tool model and a hand segmentation of the sample images, shown in Table III. Here, we use the information retrieval metrics recall, precision and the probability of error, PE [START_REF] Lee | Performance study of several global thresholding techniques for segmentation[END_REF], defined in terms of true positives, T P , true negatives, T N , false positives, F P , and

P E = (T P + F N ) F N + (T N + F P ) F P (10)
where we consider pixels labelled as the target object to be positive examples (and background negative), and the hand segmentation to be the ground truth. The F-measure is intended to give a sense of "overall" accuracy by taking into account both precision and recall, since either one alone can be trivially driven to 1 by assigning everything to background or to foreground respectively.

A. Retinal surgery

We first present results of the algorithm performing rigid tool tracking on video from a retinal eye surgery testbed. In this case, stereo microscope images of a pair of forceps against an eye phantom background are captured. The phantom is the interior of a half-sphere, painted to resemble the retinal surface.

The main challenge of this domain comes from the optics of the microscope. Due to the extreme magnification, the depth-of-field is very small, so it is impossible to keep the entire scene in focus. As a result, most of the tool shaft is blurred, making edge-based tracking challenging if not impossible. Additionally, this effect makes it difficult to get a good stereo calibration of the system. For this reason, tracking in this domain was performed on monocular video.

We perform tracking in this sequence using a 2-D mask taken from a hand-segmentation of a single frame of the video. The pose space consists of 2-D rotation and translation of this mask. Although the tool is not actually rigid, since the fingers of the forceps are articulated, we model it as such, and it remains mostly rigid within this video sequence. Sample images are shown in Figure 3.

In this case, the probabilities in Equation 1 are obtained from histogram matching. During training, a histogram h C is built for each class, C, in the LDA space in the usual fashion, and normalized to sum to 1. Then, during classification, the class probability of a pixel is taken as

P (v | C) = h C (u(πv)) (11)
where u(v) is the histogram binning function and π is the linear dimensional reduction given by LDA. The features input to the LDA computation were RGB and HSV color of the pixel, average intensity within a small window, and 5 Laplacian of Gaussian filters of different bandwidths to add texture information. Finally, the minimum and maximum values over the vector comprising these features were added as two more features.

Given the simplicity of the problem, we were able to implement the tracking algorithm in Matlab, using an optimization which iteratively searched sucessive 2D rotations and translations of the mask. This allowed us to compare the tracking results using a single model and with those obtained when performing model updating on each frame. The result were nearly identical, suggesting the algorithm is reasonably robust to moderate changes in appearance.

B. Laparoscopic surgery

Here, we track two needle driver tools of Intuitive's da Vinci robotic system [START_REF]I. Intuitive Surgical[END_REF]. The tools consist of a long shaft, followed by a wrist joint, which is connected to two grippers that can move independently. We also allowed the base of the shaft to rotate and translate freely giving a total of 9 degrees of freedom per tool. We simultaneously track two of these tools to test performance on tracking articulated objects. Thus, the final configuration space consists of 18 degrees of freedom.

Challenges of this domain include extensive specular reflections, a cluttered and changing background, and tools which change appearance as they rotate or become covered with body fluids.

Here we chose to use Gaussin mixture models (GMMs) to represent the appearance of the tool along the lines of [START_REF] Lu | A Three Tiered Approach for Articulated Object Action Modeling and Recognition[END_REF]. Both LDA and GMM computations are performed by LNKnet [START_REF] Kukolich | LNKnet User's Guide[END_REF]. Once trained, the class-conditional probabilities needed for Equation 1 are evaluated as usual [START_REF] Xu | On convergence properties of the em algorithm for gaussian mixtures[END_REF], as a function of the means, µ i , covariances, Σ i , and weighting coefficients, α i , of the C n mixture components of class C, with i ∈ {1, 2, ...C n }, and with π once again representing the LDA transformation:

P (v | C) = C n i=1 α i P (πv | µ i , Σ i ) (12)
where

P (πv | µ i , Σ i ) = 1 √ 2π|Σ i | 1 2 e -1 2 (πv-µi) T Σ -1 i (πv-µi) (13)
The particular features used are the average color value of a 3-by-3 block of pixels centered at the current location, represented in both RGB and a modified version of HSV, ConeHSV, which is defined with respect to the standard components of HSV (H, S, and V ) as

ConeHSV 1 = V (14) ConeHSV 2 = Scos(H) (15) ConeHSV 3 = Ssin(H) (16)
The effect is to reshape the standard HSV space to the conical representation sometimes used for color pickers, in which Euclidean distances are more valid. To these six features, four Haralick texture features [START_REF] Haralick | Computer and Robot Vision[END_REF] are added (contrast, correlation, energy, and homogeneity), which are based on graylevel co-occurrence matrices built from a 7-x-7 neighborhood about the current location. The final feature vector is of total length 10 with each entry ranged 0 to 1. One could choose to use a set of features tailored to the specific domain in which tracking is being performed. For simplicity and generality, we use this same set of features in each tracking domain, as it provides two complementary color descriptors as well as a rich but compact set of texture features, although Gabor filters would probably be a more suitable texture feature choice for a GPU implementation. 1) Simulated Data: We first tested the algorithm on a straightforward simulated problem. A simulated data set was generated by rendering the da Vinci tool models undergoing some translation and articulation against a black background. This sequence effectively bypasses the class probability estimation step, giving the case of an ideal segmentation and perfect object model. The ground truth configuration of the object is also available in each frame. Error was evaluated by two metrics, average Euclidean distance in the configuration space of joint angles and translations, and the image overlap metric used for the real sequences. Sample images are shown in Figure 4. As can be seen in Table III, the final configuration error was 0.01 which is at the convergence resolution of the optimization.

2) Real Data: We conducted tracking trials on a stereo video sequence of a suturing task in porcine surgery. We tested two cases: 1) a single foreground class and 2) two foreground classes. Sample images of both are shown in Figure 5. In the latter case, the needle-driver was initially modeled as 3 separate classes: shaft, wrist, and fingers, as illustrated in Figure 2. Wrist and finger pixels were not reliably distinguished though, so they were merged into a single end-effector class used in the results shown.

One can see that the multi-class case displays more reliable tracking of the end-effector. This is not surprising, as the LDA reduction we currently use would have difficulty finding a single projection that distinguishes both from the background. The precision of the method was nearly as good as the simulated sequence; however the recall is much lower, mainly due to the tool being not completely detected in the class probability estimation step.

C. Hand Tracking

We also demonstrate a hand-tracking task on a short mono video of an arm moving against a static background, in order to show the versatility of the method across different domains. Our model is quite rudimentary: each finger is modeled as a rigid cylinder with rounded ends and two degrees of rotational freedom. The wrist also has two degrees of rotational freedom. With translation of the base of the forearm, there are a total of fifteen degrees of freedom in our model, much less than the estimated 27 of the real hand [START_REF] Dewaele | Hand motion from 3d point trajectories and a smooth surface model[END_REF]. The same appearance model was used as in the laparoscopic surgery application in Section IV-B.

Sample images are shown in Figure 6. In particular we can see in frames 11 and 21 that the optimization appears to have chosen a local minimum that could be easily improved by slightly moving the thumb and forearm. Again, precision is relatively high, while recall is somewhat lower. In this case, this seems most likely due to inaccuracy in the 3D model at the edges causing a more conservative fit to be more optimal.

V. DISCUSSION

The work we have presented here is a promising first step toward a general-purpose and efficient methodology for tracking articulated objects. What is particularly compelling is that, even without sophisticated search algorithms and time-series filtering, the algorithms are able to track highly articulated surgical tools in challenging real-world situations, and operate with an image-domain accuracy that is acceptable for many applications. Most importantly, the algorithms were applied with minimal changes in three different domains with very different articulated models.

There are several immediate extensions that are clearly needed to improve the accuracy and overall performance of the method. As noted in Section III-B, then entire evaluation of the likelihood function can be implemented on the GPU, providing several orders of magnitude reduction in execution time. This in turn will make it possible to examine a large family of sampling-based search algorithms, particularly stochastic gradient methods and particle filters, with very large numbers of samples.

The generation and use of the class conditional probability maps also has many potential improvements. Making use of boundary information on tools, and unique image features (potentially registered to the underlying CAD model) are likely to improve both accuracy and robustness.

Although an approach relying entirely on edge features is not likely to fare well in the laparoscopic surgery do-Fig. 5. Images from the da Vinci laparoscopic surgery domain, demonstrating articulated tool tracking: The top row shows input frames 25, 75, 125, and 175 from the left channel of video. The middle and bottom rows show the corresponding tracking output for a single foreground class and two foreground classes respectively. The color-scheme for the middle row is the same as in previous figures: the blue channel displays the input image intensity, the red channel shows the probability of tool, and the green channel shows the the tracked tool. In the bottom row, the red channel does not show foreground probability, but instead a value corresponding to the most probable class: zero for background, half intensity for tool shaft, and full intensity for tool end effector. main, our method would likely benefit from the addition of contour information to the objective function. It would be relatively simple to render a wireframe of the object and add an additional conditional probability term to the objective function, measuring distance between the projected model contours and detected image contours, similar to that used in [START_REF] Drummond | Real-time visual tracking of complex structures[END_REF]. A robust estimator of some sort would be necessary, though, to avoid the function being dominated by spurious edge detections when they can not be detected reliably, or to de-emphasize the contribution of the edge term before gross alignment has been achieved.

Finally, the need for a small number of hand-segmented images is still a limitation of the current methods. However, we have performed preliminary experiments, not described here, using Expectation-Maximization methods to learn appearance models without hand training. The results are quite positive, but are difficult to generalize in our current implementation. Although our current model deals well with many of the domain challenges, effects like extreme changes in lighting or the covering of the target with blood or other fluids would require a self-updating model for tracking over long sequences in practical applications. More generally, non-parametric foreground models and related methods for model updating such as those described in [START_REF] Lu | A nonparametric treatment for location/segmentation based visual tracking[END_REF] are clearly needed in order to move to objects with changing appearance.

VI. ACKNOWLEDGMENTS

Fig. 1 .

 1 Fig. 1. Typical edge detection results on an image from the laparoscopic surgery domain. The input is the second image from Figure 2. On the left are two parameter settings for the Sobel filter and on the right two settings for the Canny detector. In all cases, the tool shaft is not well-detected, and many edges are detected in the background in the vicinity of the tool tip.

Fig. 2 .

 2 Fig.2. Hand-segmented images from the da Vinci laparoscopic surgery domain used to train appearance models. In the first row, a single foreground class is used and is labelled green. The second row shows 3 foreground classes: green is tool shaft, cyan is wrist, and blue is finger.

Fig. 3 .

 3 Fig. 3. Images from the retinal microsurgery domain, demonstrating rigid tool tracking: top left is the first input image of the video, then the tracking results for frames 1 and 17. On the second row are tracking results from frames 33, 49, and 65. For the tracking result images, the blue channel displays the input image intensity, the red channel shows the probability of tool output by the classifier, and the green channel shows the rendered tool model in the current belief configuration.

Fig. 4 .

 4 Fig. 4. Images from the simulated da Vinci laparoscopic surgery sequence: Frames 1, 25, 50, 75, and 100 of the left channel of the tracked sequence are shown. The color-scheme is the same as in Figure 3, though in this case the red and blue channels are identical.

Fig. 6 .

 6 Fig. 6. Hand tracking results: top left is the first input image of the video, then the tracking results for frames 1 and 11. On the second row are tracking results from frames 21, 31, and 41. The color scheme is the same as above.

TABLE I THE

 I RENDERED IMAGE WITH A PROBABILITY MAP, AND IMAGE SUM IS SUMMATION OF ALL ELEMENTS IN AN IMAGE. NUM. OPS. GIVES THE NUMBER OF TIMES EACH OPERATION MUST BE PERFORMED ONA FRAME OF VIDEO, WITH "MAX" MEANING AS MANY AS POSSIBLE IN THE TIME AVAILABLE. TOTAL GIVES THE OVERALL AMOUNT OF TIME SPENT ON THAT OPERATION EACH FRAME.

	Operation	Time/Op (ms) Num. Ops.	Total (ms)
	11x11 filter	3.8	4	15.2
	3x3 filter	0.16	4	0.64
	Probability	1	1	1
	Render	0.1	max	max
	Image Mask	0.1	max	max
	Image Sum	0.3	max	max

APPROXIMATE TIME REQUIRED TO PERFORM WHOLE-IMAGE (640-X-480) OPERATIONS ON OUR TEST SYSTEM GPU (GEFORCE 8600GTS). PROBABILITY REFERS TO COMPUTING A CLASS PROBABILITY MAP AND CONSISTS OF AN LDA PROJECTION AND TABLE LOOKUPS. RENDER REFERS TO GENERATING AN IMAGE OF THE OBJECT MODEL. IMAGE MASK IS AN ELEMENT-WISE MULTIPLICATION OF A

TABLE II NUMBER

 II OF COMPUTATIONS OF THE OBJECTIVE FUNCTION EXPECTED TO BE POSSIBLE AT EACH FRAME-RATE. EACH DEVICE IS AN NVIDIA GEFORCE MODEL. 8600GTS IS THE TEST MACHINE, AND THE OTHER TWO ARE EXTRAPOLATED VALUES BASED ON HARDWARE DIFFERENCES.

	Device	30 fps 15 fps 10 fps 5 fps	2 fps
	8600GTS	33	100	166	366	966
	8800GTX	233	380	584	1291	3515
	2x8800GTX	500	913	1384	2891	7515

We thank the National Science Foundation (NSF), for their support of this work through grants IIS-0205318 and R21 EY016359, and our reviewers, for their comments. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF.