Impaired imipenem uptake associated with alterations in outer membrane proteins and lipopolysaccharides in imipenem-resistant Shigella dysenteriae

Anindya Sundar Ghosh, Alak Kanti Kar and Manikuntala Kundu*

Department of Chemistry, Bose Institute, 93/1 APC Road, Calcutta 700 009, India


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Three imipenem-resistant mutants were obtained from a clinical isolate (C152) of Shigella dysenteriae by selection with increasing concentrations of imipenem. Resistance to imipenem was associated with resistance to several other ß-lactam antibiotics. The penicillin-binding protein (PBP) patterns of the resistant and the wild-type strains were comparable. The permeability of the outer membrane proteins (OMPs) of the most resistant mutant, IM16, was lower than that of the parent strain C152 when imipenem and arabinose were used as test solutes. This mutant had lower levels of both the major OMPs of Mr 43,000 and 38,000. There were also differences in the patterns of lipopolysaccharide (LPS) of the mutants and the wild-type strain. The mutant IM16 had less short-chain LPS than the parent C152. Increasing imipenem resistance was also associated with a concomitant decrease in the level of 2-keto-3-deoxyoctonate, a component of the core region of LPS.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Shigellae are a major cause of diarrhoeal diseases in developing countries. Strains multiply resistant to ampicillin, co-trimoxazole, streptomycin, chloramphenicol and tetracycline are a cause of growing concern. 1,2 The carbapenems are used as the last line of drugs for combating organisms such as Klebsiella pneumoniae, because of their stability against ß-lactamases. However, with the increasing use of imipenem, resistance to carbapenems is steadily emerging as a new threat to antibacterial chemotherapy. 3 Imipenem-resistant clinical isolates of Pseudomonas aeruginosa emerged at relatively high frequencies soon after the introduction of imipenem in the clinical setting, and are usually associated with the loss of an imipenem-specific porin, D2. 4 In enterobacteria, the presence of an imipenem-specific channel is yet to be demonstrated. Enterobacter cloacae strains resistant to imipenem and deficient in non-specific porins have been described. 5 Studies in our laboratory have shown that a clinical isolate of Shigella dysenteriae which lacks the major non-specific porin of Mr 43,000 retains imipenem-sensitivity while becoming resistant to a number of other ß-lactams. 6 In the present paper we have now investigated the possible mechanisms of development of imipenem resistance on exposure of S. dysenteriae to this antibiotic, since the imipenem-resistant phenotype has not been studied in S. dysenteriae.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals

Imipenem was a gift from Professor J. M. Frere, University of Liege, Belgium. Cefoperazone, cephaloridine, latamoxef, benzylpenicillin, ampicillin and amoxycillin were from Sigma Chemical Co., St Louis, MO, USA. Ciprofloxacin was a gift from Ranbaxy Laboratories, Delhi, India. Gentamicin was obtained from Nicholas Piramal India Ltd. Bombay, India. Nitrocefin was a product of BBL Micro biology Systems, Cockeysville, MD, USA. Sodium lauroyl sarcosinate, lithium dodecyl sulphate (LDS), cetyltrimethylammonium bromide (CTAB), egg phosphatidylcholine, dicetyl phosphate. 3-deoxy-D-mannooctulosonic acid (KDO), arabinose, glucose, glycine and N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate (Zwittergent (3-12)), were products of Sigma Chemical Co.

Growth of the organisms and determination of MICs

S. dysenteriae C152, a clinical isolate, was obtained from the School of Tropical Medicine, Calcutta, India. Bacteria were routinely grown in Tryptic Soybroth (Difco, Detroit, MI, USA) on a rotary shaker at a temperature of 37°C. MICs were determined on Mueller–Hinton agar plates containing serial dilutions of antibiotics by inoculating with 106 cfu and reading after 18 h of growth at 37°C.

Isolation of imipenem-resistant mutants

Imipenem-resistant mutants were isolated by selection with imipenem. One hundred microlitres of an overnight culture of strain C152 was inoculated on a 20 mL Mueller– Hinton agar plate containing imipenem at a concentration equal to its MIC for C152. After incubation for 48 h at 37°C, colonies were transferred to plates containing the same concentration of antibiotic. After repeating this procedure three times, colonies were transferred to the next two-fold higher concentration of imipenem and the entire procedure was repeated for several generations. Colonies that grew stably at a particular concentration of imipenem for several generations were chosen for further studies.

Preparation of membranes and penicillin-binding protein (PBP) assays

Cells were harvested, washed with 50 mM Tris–HCl, 5 mM EDTA (pH 8) containing 1 mg/L DNase and disrupted by sonication. After removal of undisrupted cells and cell debris, membranes were pelleted by centrifugation at 16,000g for 30 min as described previously. 7 PBP assays and competition experiments were performed on membrane proteins as described by Mahapatra et al. 7

ß-Lactamase assay

The ß-lactamase activity of sonic extracts of exponentially growing cells was routinely assayed spectrophotometrically at 482 nm with the chromogenic cephalosporin nitrocefin. 8 The hydrolysis of imipenem was assayed spectrophotometrically 9 at a wavelength (300 nm) that gave a maximum in the difference spectrum of the hydrolysed antibiotic against the unhydrolysed one ({Delta}{epsilon} = 8000 M-1 cm-1). Induction of ß-lactamase 10 was studied by adding imipenem at concentrations of 0.125, 0.25 and 0.5 mg/L to exponentially growing broth cultures, followed by withdrawal of aliquots at intervals of 15 min up to stationary phase. 11 Cells were harvested from each aliquot and ß-lactamase activity was assayed in the sonic extracts.

Preparation of outer membranes

Outer membranes were prepared by treatment of membranes with 2% (w/v) sarkosyl for 60 min at 30°C followed by centrifugation at 100,000g for 30 min. The pellet, which contained the outer membrane proteins (OMPs), was washed twice with 0.5% (w/v) sarkosyl and stored at -70°C.

Purification of the 38 kDa outer membrane protein

Outer membranes from strain M2, which lacked the 43 kDa porin, 6 were first extracted with 2% Zwittergent (3-12) for 1 h at 30°C, followed by centrifugation at 100,000g for 40 min. The pellet was then extracted with 2% (w/v) LDS for 1 h at 30°C, followed by centrifugation at 100,000g for 1 h. The supernatant was dialysed overnight against Buffer B (20 mM Tris–HCl, pH 8, 5 mM EDTA, 0.1% LDS). One milligram of protein was loaded on to a Superdex HR5/30 (10 mL) gel filtration column (Pharmacia) coupled to an FPLC system and equilibrated with Buffer B. Proteins were eluted with Buffer B and analysed by SDS–PAGE. Fractions containing only the 38 kDa protein were pooled and stored at -20°C.

Liposome swelling assay

A lipsome swelling assay was carried out according to Nikaido & Rosenberg. 11 Egg phosphatidylcholine (2.5 µmol) and dicetyl phosphate (0.1 µmol) were dried as a thin film. The film was suspended in 0.2 mL of buffer in which outer membrane (100 µg of protein) was added. The mixture was sonicated and dried under vacuum. The film was reconstituted with 0.4 mL of a solution containing 10 mM Tris–HCl (pH 8) 12 and diluted in isotonic solutions of test solutes. The decrease in absorbance at 400 nm was followed as a function of time. The isotonic concentration of stachyose as well as of test solutes was determined according to Yoshimura & Nikaido. 13

Isolation of lipopolysaccharide (LPS)

Lipopolysaccharides (LPS) were prepared from acetone-dried cells by the phenol–water extraction method of Westphal & Jann. 14 Cells were harvested, washed with phosphate-buffered saline (PBS), pH 7.4, and incubated with acetone for 1 h at 37°C with vigorous shaking to extract water and phospholipids. They were then washed three times with acetone and the pellet was dried in air. A mixture of 17.5 mL of water and an equal volume of phenol, warmed to 65°C, was added to 1 g of acetone-dried cells. The mixture was incubated at 65– 70°C for 15 min with stirring and then cooled in an ice bath to allow phase separation between the aqueous and phenol-rich layers. The mixture was centrifuged at 3000g for 15 min, and the aqueous layer was aspirated and exhaustively dialysed against water. The dialysate was centrifuged to remove insoluble matter, if any, and concentrated ten-fold under reduced pressure. Nucleic acids were precipitated by addition of 0.1 mL of a 2% (w/v) solution of CTAB. The precipitate was discarded following centrifugation at 6000g for 10 min and the supernatant was lyophilized. The freeze-dried material was dissolved in a minimum volume of 0.5 M NaCl and the crude LPS was precipitated by the addition of ten volumes of ethanol. The crude LPS was dissolved in 100 mM Tris–HCl buffer containing 5 mM CaCl2, pH 7.6, and digested sequentially with DNase (10 mg/L), RNase (10 mg/L) and pronase (100 mg/L) at 42°C for 16 h to remove contaminating protein and nucleic acids. The digested material was heated in a water bath for 30 min, exhaustively dialysed against water, lyophilized and stored at -70°C.

Polyacrylamide gel electrophoresis of LPS

After electrophoresis of the LPS preparation on a 12.5% SDS–polyacrylamide 8 M urea gel, the gel was stained with silver nitrate following the method of Hitchcock & Brown. 14 Briefly, after electrophoresis, the gel was fixed in the fixing solution (25% (v/v) isopropanol, 7% (v/v) acetic acid) overnight. The fixing solution was replaced by fresh fixative containing 0.7% (w/v) periodic acid several times and then stained using ammoniacal silver nitrate for 10 min with vigorous shaking at room temperature. After washing the gel three times, for 10 min each time, the gel was immersed in formaldehyde developer solution (prepared fresh) till the bands were visible. The developing reaction was stopped by adding an aqueous solution of 7% (v/v) acetic acid, washed and dried.

Assay for KDO in LPS

This was done as described by Karkhanis et al. 16 One millilitre 0.2 N H2SO4 was added to a test tube containing 2 mg of LPS-containing material. The reaction mixture was heated at 100°C for 30 min, cooled and centrifuged at maximum speed in a clinical centrifuge for 5 min. A volume (0.5 mL) of the supernatant was transferred into another test tube; 0.25 mL of 0.04 M periodic acid in 0.125 N H2SO4 was added to this, vortexed, and allowed to stand at room temperature for 20 min. Then 0.25 mL of 2.6% sodium arsenite in 0.5 N HCl was added, vortexed, and kept until the brown colour had disappeared. Then 0.5 mL of 0.6% thiobarbituric acid (TBA) was added; the mixture was then vortexed, and was heated at 100°C for 15 min. While hot, 1 mL of dimethylsulphoxide (DMSO) was added; the mixture was then allowed to cool to room temperature and the optical density was read at 548 nm against a blank, treated as above, without KDO. The amount of KDO in the material was calculated from a standard curve prepared using known concentrations of KDO.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibilities of C152 and its imipenem-resistant mutants to different antibiotics

Imipenem-resistant mutants were obtained at a frequency of 10-8 to 10-9. Three mutants, IM4, IM8 and IM16, are discussed here. The MICs of different antibiotics for strains C152, IM4, IM8 and IM16 are shown in Table I. Resistance of the mutants was found to several ß-lactams, while no cross-resistance was observed to structurally unrelated antibiotics like ciprofloxacin and gentamicin.


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Table I. MICs of various antibiotics for S. dysenteriae strain C152 and its isogenic mutants, IM4, IM8 and IM16
 
Colony morphology

Colonies of the parental strain, C152, on agar plates, were smooth, round and discrete. All three mutants formed rough colonies with corrugated peripheral regions. These were spread like mats over the plate.

PBP patterns of the membranes and ß-lactamase activities

Alterations in PBPs may account for imipenem resistance. The PBP profiles of the parent C152 and the imipenem-resistant mutants, IM4, IM8 and IM16, were identical to that reported by Mahapatra et al. 7 Competition experiments showed that the affinities of the PBP2 (imipenem-specific target) of these strains for imipenem were also identical (ID90, i.e. the concentration of imipenem required to inhibit benzylpenicillin binding by 90%, was 5 µM in all cases). Resistance to imipenem was not likely to be mediated by alterations of PBP(s).

Imipenem resistance may also be attributed to degradation of the antibiotic by ß-lactamases. The parent strain, C152, and the three mutants had very low ß-lactamase activity (<=2 nmol of nitrocefin hydrolysed/min/mg of protein), ruling out this possibility. Hydrolysis of imipenem was not detected. Induction of ß-lactamase by imipenem was not observed.

OMPs and uptake of solutes

S. dysenteriae has two major OMPs of Mr 43,000 and 38,000. 6 The OMP patterns were identical to that reported by Kar et al. 6 However, in the most resistant mutant, IM16, the amounts of the 43 and 38 kDa OMPs were lower than in the parent strain (Figure 1). The amount of a 20 kDa OMP was also reduced in IM16.



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Figure 1. Outer membrane protein profiles of S. dysenteriae. For each strain, 50 µg total membrane protein was loaded. Lane a, IM16; lane b, C152.

 
Uptake of solutes was studied in the mutant IM16, which showed the highest degree of resistance to imipenem. Permeabilities of the sugar arabinose and the antibiotic imipenem were measured with proteoliposomes containing crude outer membranes of C152 and IM16. Relative swelling rates reflected a lower rate of permeation of both these solutes across the outer membrane of IM16 compared with C152 (Table II).


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Table II. Swelling rates of proteoliposomes reconstituted with OMPs from IM16
 
Characterization of the 38 kDa OMP

The 38 kDa OMP was purified from strain M2 (which lacked the 43 kDa porin,) 6 as described above. Strain M2 was chosen in order to avoid the presence of the 43 kDa porin as a contaminating protein. When reconstituted into proteoliposomes, the purified 38 kDa OMP behaved as a pore-forming protein. The diffusion of various uncharged molecules (saccharides) through the channel formed by this protein was tested in liposome swelling assays. The rate of diffusion was inversely related to the size of the molecule tested (Figure 2). The specific activity was defined as the change in optical density at 400 nm x 1000/min/µg protein. The specific activity for arabinose diffusion was 53, while that of the 43 kDa porin was 330. Using a number of test solutes of different size, and applying the Renkin equation, 17 the pore diameter was determined to be 1 nm. The 38 kDa porin also served as a channel for the carbapenem imipenem. The specific activity for imipenem and cephaloridine diffusion was between 8 and 11 in three different sets of experiments with three different liposome preparations. The diffusion of several poorly penetrating ß-lactams (e.g. latamoxef or piperacillin), however, could not be reliably estimated.



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Figure 2. Diffusion rates of small molecules in liposomes reconstituted with purified 38 kDa OMP. The rates are expressed as relative to the rate of permeation of glucose, which is set at 100. The following compounds were used (from left to right): glycine, arabinose, glucose, lactose.

 
Analysis of the LPS profile

The core region of LPS from shigellae consists of KDO and L-glycero-D-manno-heptose. 18 the KDO content decreased progressively as imipenem resistance increased (Table III). The LPS profiles of the imipenem-resistant mutants and the parent were analysed on SDS–15% polyacrylamide gels containing 50% area by periodic acid–silver staining. The imipenem-resistant mutants showed a number of alterations in the region of bands corresponding to the ` core' region of LPS (Figure 3). C152 showed four major bands (labelled a–d in Figure 3), while IM4 and IM8 showed three major bands (labelled e–g). The relative migration of the bands of C152 differed from those of IM8 and IM4, suggesting that qualitative alterations in the LPS had occurred in these strains. IM16 had only one major band in the core region. IM16 showed less short-chain LPS than C152 (lane ii, Figure 3) The observation of altered colony morphology in the imipenem-resistant mutants was consistent with the altered LPS profiles.


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Table III. KDO content of the LPS of C152, IM4, IM8 and IM16
 


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Figure 3. LPS profiles of C152 and the imipenem-resistant mutants after SDS- PAGE and silver staining. Lane i, C152; Lane ii, IM16; Lane iii, IM8, Lane iv, IM4.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ß-Lactam resistance has emerged as a problem in the chemotherapy of shigellosis. ß-Lactam resistance involves an interplay of the following: (i) elaboration of ß-lactamases; (ii) altered affinities of the PBPs for ß-lactam; (iii) decreased outer membrane permeability; and (iv) overexpression of efflux pumps. 19 Exposure of Gram-negative bacteria to third-generation cephalosporins results in a high frequency of emergence of ß-lactam-resistant mutants that produce chromosomal cephalosporinase constitutively. In the study reported here, development of ß-lactam resistance was explored in a background virtually devoid of ß-lactamase. Induction of ß-lactamase or imipenem hydrolysis by ß-lactams was not observed. There was no difference in PBP profiles of the wild-type and the imipenem-resistant mutants, or in the affinities of PBP2 of these strains for imipenem. A PBP-mediated mechanism was therefore not linked to imipenem resistance. Efflux pumps have widely been demonstrated to be responsible for resistance against structurally unrelated antibiotics. 20 Since simultaneous resistance to structurally unrelated antibiotics was not observed, this argued against the involvement of an efflux system in development of imipenem resistance in the present case. However, this work does not rule out entirely the possibility of an efflux system that operates principally on ß-lactams.

The role of membrane permeability in determining antibiotic resistance is well established. 21 Consecutive mutations leading to the emergence of imipenem resistance have been reported in a clinical strain of Enterobacter aerogenes. 22 Examination of the OMP profile had, in that instance, revealed the lack of a major IMP. In-vivo selection of porin-deficient mutants with increased resistance to expanded-spectrum cephalosporins has also been reported in other Gram-negative bacteria. 23 In our laboratory we have demonstrated ß-lactam resistance in a clinical isolate of S. dysenteriae, lacking ß-lactamase and devoid of a 43 kDa non-specific porin. 6 However, this strain, while being resistant to a number of ß-lactams, remained sensitive to imipenem. Mutant IM16, isolated by progressive exposure of a sensitive strain to increasing concentrations of imipenem, showed a decrease in the level of the 43 and 38 kDa OMPs when OMPs derived from the same amount of total membrane protein for each strain were run on SDS–polyacrylamide gels. The test solutes, arabinose and imipenem, permeated the outer membrane of IM16 more slowly, suggesting that this could be a factor accounting for imipenem resistance. Since our previous findings indicate that the absence of the 43 kDa porin was not sufficient to confer imipenem resistance, 6 it appeared possible that the reduced level of the 38 kDa porin could be one of the determining factors in imipenem resistance of S. dysenteriae. The 38 kDa porin was purified and demonstrated to be a pore-forming protein, albeit with lower activity than the 43 kDa porin. The former protein was also found to serve as a channel for imipenem. We speculate that it may serve as an alternative, although not specific, channel for imipenem in the absence of the 43 kDa porin. This accounts for the fact that the previously reported cefoxitin-resistant clinical isolate M2, lacking the 43 kDa porin, remains susceptible to imipenem.6

Other factors that could possibly alter the permeability characteristics of the mutants, were analysed. There were alterations in LPS in all the resistant strains. Estimation of KDO showed a progressive decrease with increasing imipenem resistance, and the band patterns in the core region were also different from that of the parent C152. Alterations in LPS were accompanied by a change in colony morphology to a `rough' variety in the three mutants. LPS has been identified as an important outer membrane component required for the assembly of the trimeric PhoE porin of Escherichia coli. 24 The assembly of OmpF has been reported to be less efficient in a mutant of E. coli with a defective core region. 25 Hydrogen bonding, coulombic attraction and cation-mediated bonding between the core oligosaccharide of LPS and ionogenic amino acid residues at the outer rim of the porin channel probably modulate operation of the porin channels. 26 In P. aeruginosa, it has been proposed that interaction of LPS with porins may influence the conformation of the porins and the number of open, functional pores. 27 Imipenem channels (protein D2 or Opr D2) of P. aeruginosa are mostly closed in the LPS-free membrane. 28 Alterations in porin as well as LPS have been reported to be associated with the development of resistance towards several antibiotics in Burkholderia cepacia. 29 Altered LPS patterns and reduced amounts of LPS in the resistant mutants may influence the number of available open pores of the porins of the mutants.

The higher MICs of cephaloridine, cefoperazone and latamoxef, which utilize non-specific porin channels, suggest that the channel-forming properties of the 43 kDa porin were probably altered in the mutants. An imipenem-specific channel is yet to be identified in S. dysenteriae. The isolation of imipenem-resistant mutants lacking the 38 kDa porin would strengthen the view that this protein is associated with imipenem permeability. Such mutants have not been isolated. However, our present studies demonstrate that diminished levels of both the 43 and the 38 kDa porin are necessary for manifestation of imipenem resistance. This also leads to resistance to other ß-lactams. Although it is difficult to propose any direct correlation between the development of resistance and LPS pattern, circumstantial evidence suggests that the altered LPS patterns, arising from exposure to imipenem, also affect the permeability of the mutant strains. Alterations in LPS have, in a number of instances, been linked to the antibiotic susceptibility of bacteria. 20,28

In conclusion, our studies provide evidence that alterations in the major OMPs of 43 and 38 kDa, and in LPS occur following exposure of S. dysenteriae to imipenem and confer imipenem resistance on S. dysenteriae. Simultaneous resistance to a number of other ß-lactams is also observed. The fact that exposure to imipenem may lead to the emergence of strains resistant to imipenem as well as other ß-lactams should be sufficient reason to exercise caution in the use of this antibiotic. It would be of further interest to study the effect of LPS isolated from each of these mutants in channel-forming properties of the porins of S. dysenteriae incorporated in proteoliposomes, to analyse the fatty acid profiles of the LPS, and to evaluate the role of altered OMPs and LPS in the development of imipenem resistance in clinical isolates of S. dysenteriae.


    Acknowledgments
 
This work was supported in part by grants from the Department of Science and Technology and the Council of Scientific and Industrial Research (CSIR), Government of India to M. K.; A. K. K. was supported by a fellowship from the CSIR.


    Notes
 
* Corresponding author. Fax: +91-33-3506790. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 11 May 1998; returned 29 June 1998; revised 26 August 1998; accepted 15 September 1998





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