Chromosomal ß-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa

Oana Ciofua,*, Terry J. Beveridgeb, Jagath Kadurugamuwa{dagger}, Jan Walther-Rasmussena and Niels Høibya

a Institute for Medical Microbiology and Immunology, Panum Inst. 24.1, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark; b Department of Microbiology, University of Guelph, Guelph, Canada


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Membrane vesicles were isolated from one ß-lactam-sensitive and three ß-lactam-resistant Pseudomonas aeruginosa clinical isolates from patients with cystic fibrosis. The presence of the chromosomally encoded ß-lactamase in the membrane vesicles was shown by electron microscopy and enzymatic studies. This is the first report of extracellular secretion of ß-lactamase in P. aeruginosa and it seems that the enzyme is packaged into membrane vesicles.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ß-Lactam antibiotics in combination with aminoglycosides or quinolones are the drugs of choice in patients with cystic fibrosis and chronic pulmonary infection with Pseudomonas aeruginosa.1 Unfortunately, development of resistance to these drugs is a common problem.2 Resistance to ß-lactam antibiotics is mainly due to hyperproduction of the chromosomally encoded ß-lactamase.3,4

High levels of ß-lactamase activity were found in sputum of patients with cystic fibrosis during antipseudomonal treatment. The origin of this extracellular ß-lactamase activity was initially thought to be exclusively from lysed and broken cells.5 Like many Gram-negative bacteria, P. aeruginosa releases membrane vesicles filled with periplasmic components during normal growth.6 During their release from the bacterial surface the lipopolysaccharide is exposed on the outer face of the vesicle, outer membrane proteins remain integrated in the membrane vesicle bilayer and membrane vesicles become filled with periplasmic constituents.7 For P. aeruginosa these periplasmic components include protease, proelastase, peptidoglycan hydrolase, phospholipase C and alkaline phosphatase.8,9 Membrane vesicles probably contribute to P. aeruginosa infections.7 ß-Lactamase is an enzyme located in the periplasm, where it inactivates the ß-lactam molecules before they reach their targets situated on the cytoplasmic membrane, the penicillin binding proteins.

Since membrane vesicles package periplasmic components, cystic fibrosis-derived P. aeruginosa clinical isolates frequently become resistant to ß-lactams and ß-lactamase can be found free in the lung fluid, we speculated that ß-lactamase may be a constituent of membrane vesicles.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and ß-lactamase production

The presence of chromosomally encoded ß-lactamase was assessed in the membrane vesicles of one ß-lactamsensitive (15809B) and three ß-lactam-resistant (258, 17107B, 15461) P. aeruginosa clinical isolates from patients with cystic fibrosis as well as in the membrane vesicles of a sensitive reference strain (ATCC 27853). They represented different strains as identified by pulsed field gel electrophoresis (results not shown). The MIC of ceftazidime for the P. aeruginosa strains was determined by the agar dilution method with an inoculum of 104 cfu/spot using IC 50 agar (Statens Serum Insitute, Copenhagen, Denmark).

P. aeruginosa strains were grown in basal conditions and exposed to benzyl penicillin (500 mg/L) for 2.5 h to induce production of ß-lactamase. Nitrocefin was used as a substrate to detect ß-lactamase activity in sonicated extracts, which was measured spectrophotometrically.3

Transmission electron microscopy of membrane vesicles

To isolate the membrane vesicles, the cells were grown in trypticase soy broth (1 L) (Difco, Detroit, MI, USA) at 37°C, harvested in early stationary phase by centrifugation at 6000g and the supernatant was filtered sequentially through 0.45 µm and 0.22 µm pore-size cellulose acetate membranes (MSI, Westboro, MA, USA) to remove residual cells.

Membrane vesicles were recovered from the filtrate by ultracentrifugation at 150,000g for 3 h at 5°C in a Beckman 45 Ti rotor (Beckman Instruments, Inc., Toronto, Canada). The membrane vesicle pellet was washed once with 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) buffer pH 6.8 (Research Organics, Inc., Cleveland, OH, USA), repelleted by centrifugation at 120,000g for 30 min and resuspended in 100 mM phosphate buffer pH 6.8.7

The isolated membrane vesicles from the different P. aeruginosa strains were analysed by transmission electron microscopy (TEM) after negative staining with 2% w/v uranyl acetate or thin sectioning of plastic embedded samples according to Beveridge.10 For negative staining, a 20 µL volume of purified membrane vesicles was placed on carbon- and Formvar-coated copper grids, which were then stained with 2% aqueous uranyl acetate, rinsed and examined with a Philips EM 300 transmission electron microscope operating under standard conditions at 60 kV with the cold trap in place.

To prepare the thin sections, P. aeruginosa cells or purified membrane vesicles were placed in 2% molten Noble agar (Difco), put through a mild fixation-LR (London Resins Marivac, Halifax, Nova Scotia, Canada) white embedding regimen. Figure 1(b)Go shows the membrane vesicles from strain 15461 which were typical for all other strains.




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Figure 1. (a) A thin section of isolated membrane vesicles from strain 258 which has been labelled with immunogold to detect ß-lactamase. Virtually all gold is associated with the membrane vesicles. The arrows point to some of these gold particles. Bar = 100 nm. (b) Immunogold detection of ß-lactamase on thin sections of a 15461 cell after induction with benzyl penicillin. Note that the gold particles (small arrows) are associated with the cytoplasm, the outer membrane and emerging membrane vesicles. Bar = 100 nm. The large arrow points to a cluster of membrane vesicles. For the experiments shown in (a) and (b), positive controls (using non-specific antibodies) and negative controls (using protein A–gold alone) showed little or no labelling.

 
The presence of ß-lactamase in the membrane vesicles was determined by an indirect immunogold labelling technique of thin sections using polyclonal antibodies against chromosomal ß-lactamase of P. aeruginosa and protein A–gold (Sigma, St Louis, MO, USA).10

Polyclonal anti-ß-lactamase antibodies were purified, according to Harboe and Ingild11 from rabbits immunized with 130 mg/L purified chromosomal ß-lactamase emul-sified in Freund's incomplete adjuvant per injection. The rabbits were injected with this amount of protein on day 1 and given ‘booster’ injections of the same amount after 2, 4, 6 and 8 weeks. Purification of the chromosomal ß-lactamase has been described previously.12 The polyclonal antibodies showed specificity for the chromosomally encoded ß-lactamase in crossed-immuno-electrophoresis and Western blotting with whole cell sonicate (data not shown).

Isoelectric focusing (IEF) of the membrane vesicles

The membrane vesicles were focused in a 1.4% (w/v) agarose IEF gel (250 x 110 x 0.5 mm) in 12% (w/v) sorbitol supplemented with 3% (w/v) Pharmalyte 3-10.5 (Amersham Pharmacia Biotech, Uppsala, Sweden) and 0.6% (w/v) BioLyte 7-9 (Bio-Rad, Hercules, CA, USA). The gel was focused at 1000 V and for 1500 Vh at 100°C, in an LKB Multiphor unit (Amersham Pharmacia Biotech). In order to detect the enzymatic activity in the membrane vesicles isolated from P. aeruginosa strains producing low or moderate levels of ß-lactamase, larger amounts of protein were loaded on the IEF gel compared with the membrane vesicles of high producers. The amounts of protein applied per lane were: 4 µg for 258, 6.2 µg for 17107B, 62 µg for 15809 membrane vesicles, 15 µg for 15461 and 44 µg for ATCC 27553. The sonicated extract of 17107B was used as a positive control (2 µg per lane). The ß-lactamase activity of the focused proteins was determined by overlaying a 3% molten SeaKem agar (FMC Bioproducts, Roskland, ME, USA) containing 50 mg/L nitrocefin, as described by Sanders.13 Once the agar had hardened, ß-lactamase appeared as pink bands in a yellow background. The developed gel was photographed with a Polaroid MP-4 camera with Polacolor 679 film. The pI standard (IEF-MIX 3.6–9.3, Sigma, St Louis, MO, USA) was visualized by Coomassie Blue staining.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The tableGo shows the MICs and the ß-lactamase activity of the sonicated extracts of the P. aeruginosa strains from which membrane vesicles were later isolated. The phenotypes of ß-lactamase production were: high basal, constitutive (258); moderate basal, inducible (17170B, 15461 and 15809) and low basal, inducible (ATCC 27853).14


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Table. The MIC of ceftazidime and basal/induced levels of ß-lactamase in the P. aeruginosa strains used in this study
 
The TEM analysis showed similar size (50–150 nm), shape and contour (spherical) of the membrane vesicles isolated from the different P. aeruginosa strains (data not shown).

Figure 1(a)Go shows the presence of gold-coupled anti- ß-lactamase antibodies associated with the membrane vesicles of 258 which has stable derepressed production of ß-lactamase (Table)Go. Because the thin sections are c. 600 nm thick and the membrane vesicles are so small (i.e. 50–150 nm in diameter) it is difficult to determine exactly where the ß-lactamase is located in a membrane vesicle; only the outer face of the thin section is labelled and membrane vesicles have many orientations to the outer face. Because most label is associated with the lumen of the membrane vesicles (Figure 1aGo), it is likely that the enzyme is packaged in the lumen and was a periplasmic component. Figure 1(b)Go demonstrates how whole cells were frequently labelled and presumably shows the trafficking of the ß-lactamase from the cytoplasm to the periplasmic space where it is packaged into a membrane vesicle. This is consistent with the secretion pathway of ß-lactamase which is synthesized in the cytoplasm and translocated across the plasma membrane to its periplasmic location. In the particular case of Figure 1(b)Go, the cells (strain 15461 which has inducible, partially derepressed production of ß-lactamase) had been induced for 2.5 h with benzyl penicillin.

The IEF pattern of the membrane vesicles isolated from the different P. aeruginosa strains is shown in Figure 2Go. The membrane vesicles from the different clinical isolates showed ß-lactamase activity with the major band located between a pI of 8.8 and 8.6, which is characteristic for class C ß-lactamase from P. aeruginosa.15 It also shows that the ß-lactam molecules (like nitrocefin in this case) have access to the ß-lactamase packaged in membrane vesicles. No ß-lactamase activity was present in the membrane vesicles of the reference strain P. aeruginosa ATCC 27853. The production of ß-lactamase in this strain was very low compared with the clinical isolates (Table)Go.



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Figure 2. Isoelectric focusing of membrane vesicles containing ß-lactamase using nitrocefin agar detection system. Lane 1: sonicated extract of 17107B (positive control); lane 2: 258 membrane vesicles; lane 3: 17107 membrane vesicles; lane 4: 15809 membrane vesicles; lane 5: 15461 membrane vesicles and lane 6: ATCC 27853 membrane vesicles. All membrane vesicles from the clinical isolates show ß-lactamase activity with a pI value between 8.6 and 8.8. The pI values of the standard are shown by arrows.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is the first study showing the secretion of ß-lactamase in P. aeruginosa; the implications are important. Because the lipid bilayer of a membrane vesicle is the outer membrane of the donor bacterium, the membrane vesicles are freely permeable to antibiotics so that the enzyme can inactivate the drug once it enters the membrane vesicle lumen. This should effectively lower the antibiotic concentration at infection foci.

The previous observation that the number of membrane vesicles released by P. aeruginosa is increased after exposure to gentamicin8 might have important clinical implications in the treatment of patients with cystic fibrosis infected with ß-lactam-resistant P. aeruginosa strains. The combination of a ß-lactam antibiotic with an aminoglycoside is commonly used in the anti-pseudomonal treatment of these patients.1 The treatment with aminoglycoside would increase the number of membrane vesicles loaded with ß-lactamase from P. aeruginosa strains overproducing the enzyme and the synergic effect between aminoglycosides and ß-lactam antibiotics could be changed to an antagonistic effect in the case of P. aeruginosa strains hyperproducing the enzyme. Studies analysing this effect are in progress in our laboratory.

In the lung of cystic fibrosis patients the bacteria are growing in biofilm and the presence of membrane vesicles in natural occurring biofilm has been demonstrated.16 In a mathematical model, Dibdin et al.17 showed that the penetration of ß-lactam antibiotics in a biofilm is impaired by the presence of extracellular ß-lactamase the source of which was considered to be a ‘sacrificial layer of bacteria at a surface exposed to an antibiotic’. The extracellular ß-lactamase will inactivate the antibiotic as it penetrates, thereby protecting deeper-lying cells. In the light of our findings, the biofilm in the lungs of patients with cystic fibrosis might be loaded with ß-lactamase packaged into membrane vesicles that might protect the cells located more deeply inside a microcolony.

The presence of ß-lactamase packaged in these membrane vesicles can also provide an explanation for the previous observation that a major part of the ß-lactamase in biofilms is located extracellularly (B. Giwercman, unpublished results).

This present research emphasizes the importance of the natural release of membrane vesicles from Gram-negative bacteria since it implies a key role for periplasmic ß- lactamase in membrane vesicles during general resistance to ß-lactam antibiotics at the infection site.


    Acknowledgments
 
We thank Anu Saxena, Dianne Moyles and Tina Wassermann for their excellent technical assistance in this project. The research performed in T.J.B.'s laboratory was supported by a National Centre of Excellence award through the Canadian Bacterial Disease Network. The electron microscopy was performed in the NSERC Guelph Regional STEM Facility which is partially supported by an NSERC Major Facilities Access grant to T.J.B.


    Notes
 
* Corresponding author. Tel: +45-3532-7899; Fax: +45-3532-7693; E-mail: O.Ciofu{at}immi.ku.dk Back

{dagger} Present address. ROCHE Vitamins Inc., Research and Development, Building 102, B309, 340 Kingsley Street, Nutley, NJ 07110-1199, USA. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Høiby, N. (1992). Prevention and treatment of the infections in cystic fibrosis. International Journal of Antimicrobial Agents 1, 229–38.

2 . Ciofu, O., Giwercman, B., Pedersen, S. S. & Høiby, N. (1994). Development of antibiotic resistance in Pseudomonas aeruginosa during two decades of antipseudomonal treatment at the Danish CF Centre. Acta Pathologica, Microbiologica, et Immunologica Scandinavica 102, 674–80.

3 . Giwercman, B., Lambert, P., Rosdahl, V. T., Shand, G. H. & Høiby, N. (1990). Rapid emergence of resistance in Pseudomonas aeruginosa in cystic fibrosis patients due to in-vivo selection of stable partially derepressed ß-lactamase producing strains. Journal of Antimicrobial Chemotherapy 26, 247–59.[Abstract]

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Received 2 July 1999; returned 24 August 1999; revised 1 September 1999; accepted 16 September 1999