UMR 6522 CNRS, IFRMP 23, Faculté des Sciences de Rouen, 76821 Mont-Saint-Aignan Cedex, France
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Abstract |
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Introduction |
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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 glucosesalt 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.
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Materials and methods |
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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: TrisNH2, 0.6 g; TrisHCl, 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 (SigmaAldrich, St Louis, MO, USA) was determined by a standard broth microdilution technique using MuellerHinton 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 SDSPAGE, 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 (SigmaAldrich). 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 SDSPAGE, 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 methanol10% 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.
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Results and discussion |
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The conventional MIC of latamoxef for suspended E. coli cells was 6.4 mg/L. However, timekill experiments revealed the resistance of suspended E. coli cells to latamoxef 128 mg/L when incubated in LPM (Table 3). 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 2
). 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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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 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.
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Notes |
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References |
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Received 17 May 2001; returned 24 July 2001; revised 17 September 2001; accepted 8 October 2001