Phosphate deprivation is associated with high resistance to latamoxef of gel-entrapped, sessile-like Escherichia coli cells

Sébastien Vilain, Pascal Cosette, Guy-Alain Junter and Thierry Jouenne,*

UMR 6522 CNRS, IFRMP 23, Faculté des Sciences de Rouen, 76821 Mont-Saint-Aignan Cedex, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Viable Escherichia coli cells were entrapped in agar gel layers and incubated in a phosphate-limited glucose medium. Immobilized bacteria displayed enhanced alkaline phosphatase activity and overexpressed the outer membrane protein PhoE as compared with free-floating organisms. These observations highlighted the existence of high phosphate deprivation within biofilm-like structures. In addition, the antimicrobial efficacy of latamoxef against immobilized bacteria was partly recovered in the presence of a high phosphate concentration. From these data, a possible role of phosphate deprivation in the high resistance of sessile-like organisms to antibiotics may be considered.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
It has now been established that bacteria in nature have a marked tendency to adhere to surfaces and initiate biofilm formation.1 In the medical area, biofilms are responsible for dental plaque, and persistent infections on medical implants and in the lungs of patients with cystic fibrosis.2 A striking feature of biofilm bacteria is their high resistance to stresses compared with their free-floating counterparts. In particular, the lower susceptibility of fixed organisms to antimicrobial agents (antibiotics, biocides) has been extensively investigated.3 However, the complex mechanisms involved in this resistance are currently not well understood.4

We previously presented an in vitro model structure of biofilms that consists of viable bacterial cells immobilized in an agar gel layer.5 Like bacteria in natural biofilms, gel-entrapped organisms offered enhanced resistance to antibiotics.5,6 Inorganic phosphate is likely to be present at low concentrations in physiological fluids where biofilms develop.7 The purpose of the present paper was to investigate the role of phosphate deprivation in the low susceptibility of gel-entrapped, sessile-like organisms to antimicrobial agents. Free-floating and immobilized Escherichia coli cells were grown in a phosphate-limited glucose–salt medium. We then compared the alkaline phosphatase (Apase) activity and PhoE porin expression in free and entrapped bacteria, and tested their susceptibility to latamoxef in the phosphate-limited medium.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strain and inoculum preparation

A wild strain of E. coli (strain 80990) was provided by the Microbiology Laboratory of Charles Nicolle Hospital in Rouen, France. Bacteria were grown at 37°C in Mueller– Hinton medium (Difco Laboratories, Detroit, MI, USA). After incubation for 18 h, the culture was centrifuged at 1300g for 15 min, then the resulting pellet was washed and resuspended in sterile distilled water. Populations of the bacterial suspensions were estimated by optical density measurements at 546 nm referred to a calibration curve.

Preparation and incubation of immobilized cell structures

A 2% (w/v) solution of agar (Diagnostics Pasteur, Marnes-la-Coquette, France) in sterile distilled water was cooled to 38°C and mixed with a calibrated inoculum of E. coli cells (final concentration 107 cfu/mL gel). After homogenization, a volume of 20 mL of the mixture was poured into a Petri dish where it hardened at room temperature. A 25 mL volume of growth medium was then poured on the cell-loaded agar plate. The dishes were incubated for 48 h at 37°C in very high (VHPM), high (HPM) or low (LPM) phosphate medium, which was replaced every 12 h. LPM had the following composition per litre of distilled water: Tris–NH2, 0.6 g; Tris–HCl, 15 g; NH4Cl, 0.5 g; CaCl2, 0.05 g; MgSO4.7H2O, 0.05 g; FeSO4.7H2O, 0.005 g; MnSO4.H2O, 0.005 g; glucose, 15 g; yeast extract (Difco), 2 g. In LPM, inorganic phosphate (Pi) is only provided by yeast extract (final concentration in the medium c. 1 mM). In VHPM and HPM, phosphate buffer (pH 7) was substituted for Tris buffer. VHPM contained 0.5 M Pi (KH2PO4, 68.05 g/L; K2HPO4, 87.1 g/L), whereas HPM contained 0.1 M Pi (KH2PO4, 13.61 g/L; K2HPO4, 17.42 g/L).

MIC determination

The MIC of latamoxef (Sigma–Aldrich, St Louis, MO, USA) was determined by a standard broth microdilution technique using Mueller–Hinton medium (Difco) and inocula of 107 cfu/mL. The MIC was defined as the lowest antibiotic concentration inhibiting visible growth after incubation at 37°C for 24 h.

Susceptibility tests

At the end of the 48 h incubation period in LPM, HPM or VHPM, the immobilized cell structures (i.e. 2 day old cell-loaded agar plates) were rinsed with sterile distilled water. A 25 mL volume of LPM, HPM or VHPM supplied with a bactericidal concentration (20 x MIC) of antibiotic, i.e. 128 mg/L, was poured into the dishes containing the immobilized cell structures. Dishes were then incubated for 1 day at 37°C. A set of 21 identical dishes was necessary for each experiment. Every 2 h during the 1 day incubation step, one of the immobilized cell structures was removed from its dish, rinsed with sterile distilled water and then homogenized in 15 mL of distilled water using a blender. Appropriate dilutions of the resulting mixture were plated out on Plate Count Agar plates (Difco). Colonies were enumerated after incubation of the plates for 24 h at 37°C. All counts were performed in duplicate.

Killing assays were also performed on free-cell cultures (initial cell content c. 109 cfu/mL) in 50 mL Erlenmeyer flasks containing 15 mL of LPM or HPM supplemented with latamoxef. Suspended cultures (see inoculum preparation above) were inoculated in flasks containing either LPM, HPM or VHPM medium and cultured for 48 h at 37°C. Bacteria in flasks where growth had occurred were collected by centrifugation at 1300g for 15 min and resuspended in the medium used for the preceding culture step. These suspensions were used as inocula for free-cell killing assays. Suspended cultures were exposed to the same inhibitor concentration as immobilized cells. Control growth of immobilized and suspended cultures was also monitored in the absence of antibiotic.

Preparation and analysis of outer membrane extracts

After immobilized-cell gel discs had been homogenized, gel particles were eliminated by filtration through a glass-fibre membrane (GF/C from Whatman, Maidstone, UK) in a vacuum. Bacteria present in the filtrate were collected by centrifugation at 1300g for 15 min and the pellet was resuspended in sterile distilled water. Crude outer membrane extracts were prepared from bacterial pellets and analysed by SDS–PAGE, as described previously.8 The same procedure was followed for free-cell cultures.

Alkaline phosphatase (Apase) assays

Spheroplasts collected during the preparation of outer membrane extracts were resuspended in a lysis buffer of the following composition: Tris, 50 mM; NaCl, 0.5 M; EDTA, 10 mM; PMSF, 1 mM; DTT 1 mM; glycerol, 50 g/L; Triton X-100, 10 g/L). Next they were disrupted by sonic treatment at 4°C using an ultrasonicator (20 W at 5 s intervals for 8 min). The cell debris was sedimented by centrifugation at 10 000g for 15 min and the supernatant was used as the source of Apase. Protein contents were determined according to Lowry et al.9

Apase activity was determined spectrophotometrically. The reaction mixture, the temperature of which was maintained at 37°C, consisted of 800 µL of 0.1 M Tris (pH 8.8) and 500 µL of 15 mM p-nitrophenyl phosphate (PNPP). Optical density at 410 nm was measured 24 h after the addition of 200 µL of crude Apase solution (protein extract). The drop in optical density was related to the number of enzymic units using a calibration curve obtained with commercial Apase (Sigma–Aldrich). One unit of Apase activity was defined as the amount of enzyme that hydrolyses 1 µM of PNPP (pH 10.4) in 1 min at 37°C.

Apase activity in immobilized bacteria was evaluated at different locations of the cells within the polymer matrix. Each gel disc was cut into three slices (1 mm thick) using a microtome.5 Bacteria were removed from the gel as described above and the Apase activity measured in the three gel portions.

N-terminal amino acid sequence analysis

After SDS–PAGE, proteins were electrotransferred on to PVDF membrane (Millipore, Freehold, NJ, USA). Protein bands were visualized by staining with 0.1% w/v Coomassie Brilliant Blue R 250 (Sigma). The relevant protein band was excised, destained in 40% v/v methanol–10% v/v acetic acid for a few minutes, rinsed with water and air dried. The N-terminal sequence of the protein was determined by introducing the blot into an Applied Biosystems 492 automated protein sequencer (Applied Biosystems, Cortabeuf, France). A run of Edman degradation (18 cycles of pulsed-liquid chemistry) was carried out. The sequence obtained was matched to public protein sequence databases with the BLAST algorithm.10

Data analysis

All experiments were performed at least in triplicate. Results were expressed as mean ± standard deviation (s.d.). Calculations were performed using scientific graphic software (Sigmaplot from Jandel Scientific, Corte Madera, CA, USA). Results were analysed using Student's t-test. Differences were considered significant if the P value was <=0.05.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Apase is usually produced by bacteria in the absence of Pi and represents a good indicator of Pi deprivation.11 When grown in LPM, free E. coli cells displayed a low constitutive level of Apase activity, which did not significantly differ from that expressed by cells cultured in HPM (P > 0.1) (Table 1Go). The Apase activity in organisms from biofilm-like structures that had been incubated in LPM was significantly higher than that obtained in free counterparts (P <= 0.05). No significant increase in enzymic activity was observed for artificial biofilms grown in HPM (P > 0.1), whereas incubation in VHPM induced the quasi-disappearance of Apase activity in immobilized cells. The spatial distribution of Apase activity in the gel layer was relatively homogeneous, the enzyme activity slightly decreasing from the upper to the lower gel area (Table 2Go). This is consistent with observations reported by Huang et al.,12 who showed using fluorescence staining that Apase activity was distributed quasi-uniformly throughout Klebsiella pneumoniae biofilms grown for 24 h in a LPM.


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Table 1. Comparison of Apase activity in free and immobilized E. coli cells
 

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Table 2. Spatial distribution of Apase activity in a biofilm-like structure incubated in LPM
 
The SDS–PAGE analysis of the outer membrane proteins (Figure 1Go) revealed the overexpression of a protein band with an apparent mol. wt of 39 kDa in gel-entrapped E. coli cells incubated in LPM. This protein band was absent from electropherograms corresponding to free-cell cultures in LMP and immobilized-cell cultures in either HPM or VHPM. The N-terminal amino acid sequence analysis of this protein showed that it displayed 100% homology with the outer membrane protein PhoE. PhoE is an anion-selective porin known to be produced under phosphate limitation.13,14 It has been shown that PhoE is not induced by nitrate or glucose limitation,15 which suggests that induction of this protein is specific for phosphate limitation rather than being simply the result of a slow growth rate. In addition to PhoE, immobilized cells incubated in VHPM did not express a protein band of 37 kDa previously identified as porins OmpC and OmpF.8 This disappearance is questionable, since it is well known that OmpF plays a predominant role in the diffusion of ß-lactam antibiotics.16 It might reflect the existence of some other pathways for antibiotic penetration into microorganisms.



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Figure 1. SDS–PAGE analysis of the outer membrane proteins of suspended or immobilized E. coli cells. Lane Mr, mol. wt markers; lane 1, free cells incubated in LPM; lane 2, gel-entrapped organisms incubated in LPM; lane 3, gel-entrapped organisms incubated in HPM; lane 4, gel-entrapped organisms incubated in VHPM.

 
The high Apase activity and the overexpression of PhoE in immobilized cells highlighted the existence of high phosphate deprivation within biofilm-like structures incubated in a LPM. Such environmental conditions are commonly found in the body fluids where natural biofilms develop, e.g. in plasma where concentrations in the region of 1 mM of HPO42– are found.7 This low phosphate availability to gel-entrapped bacteria is very probably due to mass transfer limitation in the polymer matrix. Hindered diffusion of solutes in biofilms17 and artificial immobilized cell structures18 is well known.

The conventional MIC of latamoxef for suspended E. coli cells was 6.4 mg/L. However, time–kill experiments revealed the resistance of suspended E. coli cells to latamoxef 128 mg/L when incubated in LPM (Table 3Go). The exposure of bacteria for 24 h to antibiotic-containing LMP reduced the number of culturable microorganisms to 24% of the initial cell population, whereas the antibiotic concentration was bactericidal (5 log kill in 6 h) in HPM. Measurements of bacterial growth rates in LMP and HPM demonstrated that the generation time was not modified by the change in Pi content of the broth: values of 22 and 23 min were obtained in LMP and HPM, respectively (Figure 2Go). These data showed that phosphate starvation induced a high resistance of bacteria to latamoxef that was independent of the growth rate. This is quite inconsistent with the suggestion made by Van Gelder et al.19 who postulated that anionic antibiotics use the PhoE channel to penetrate into Gram-negative bacteria.


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Table 3. Antibacterial activity of latamoxef 128 mg/L against E. coli cells cultivated as suspensions in LPM and HPM
 


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Figure 2. Growth curves of free-cell cultures of E. coli in LPM ({blacksquare}), HPM (•) and VHPM ({diamondsuit}). S.DS (n = 3) remained <1% of the experimental values and bars are not visible.

 
Gel-entrapped bacteria were resistant to latamoxef when incubated in either LPM or HPM (Table 4Go). After exposure for 24 h to latamoxef 128 mg/L, the number of cultivable cells did not decrease for sessile-like bacteria incubated in LPM, and c. 16% of the initial cell population was recovered in HPM. Nevertheless, increasing the phosphate concentration of the culture medium to 0.5 M allowed recovery of part of the latamoxef activity, since the immobilized cell population decreased by about 3 log units after treatment for 24 h (Table 4Go). The delayed killing effect may be due to diffusion limitations of phosphate and/or latamoxef within the immobilized cell structure, although we showed that the diffusion of latamoxef in the gel structure was moderately restricted (i.e. decreased by c. 30%).5


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Table 4. Antibacterial activity of latamoxef 128 mg/L against immobilized E. coli cells cultivated in LPM, HPM and VHPM
 
The second main hypothesis advanced to account for the resistance of microbial biofilms to antimicrobial agents is a physiology-based explanation, attributing the reduced susceptibility of biofilm organisms compared with their free counterparts to physiological differences between the bacteria in these two modes of growth.20 A common assumption states that nutrient and/or oxygen limitation occurring within biofilms induces a reduction in the growth rate. Slowly growing cells are particularly recalcitrant to inhibitors (see Gilbert & Brown3 and references therein). Recently, our group approached a detailed description of the physiology of artificially gel-entrapped organisms by presenting a comparative analysis of the protein patterns in free and immobilized E. coli cells, based on twodimensional gel electrophoresis.21 Although some of these changes might be attributed to the reduction in the physiological growth rate of gel-entrapped organisms, we showed that protein expression in immobilized E. coli cells was significantly different from that in stationary phase organisms.21

From the present results, it seems not unreasonable to postulate that phosphate starvation within biofilms and the molecular events that it induces22 might contribute to the extraordinarily high resistance of sessile organisms to antibiotics. This effect might involve a gene such as rpoS, which has been suggested to be important for biofilm formation.23 The alternative sigma factor {sigma}s (RpoS) is responsible for the activation of at least 30 genes.24 Gérard et al.25 reported that RpoS, LexA and H-NS global regulators were involved in the survival of E. coli under aerobic, phosphate-starvation conditions. Is rpoS in immobilized cells induced by phosphate limitation only? Probably not, as this global regulator of the general stress response can be induced by numerous stresses.26 Greenway & England27 presented results on the intrinsic resistance of rpoS defecting E. coli mutants to various antimicrobial agents. More recently, Cochran et al.28 showed that AlgT and RpoS displayed a statistically significant contribution to resistance to hydrogen peroxide of thin biofilm-like alginate beads entrapping Pseudomonas aeruginosa cells. Studies devoted to the evaluation of the role of rpoS in antibiotic resistance of sessile bacteria are currently in progress.


    Notes
 
* Corresponding author. Tel: +33-2-35-14-66-80; Fax: +33-2-35-14-67-13; E-mail: thierry.jouenne{at}univ-rouen.fr Back


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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received 17 May 2001; returned 24 July 2001; revised 17 September 2001; accepted 8 October 2001





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