Multiple Antibiotic Resistance in Pseudomonas aeruginosa: Evidence ...

Vol. 175, No. 22

JOURNAL OF BACrERIOLOGY, Nov. 1993, p. 7363-7372

0021-9193/93/227363-10$02.00/0 Copyright C) 1993, American Society for Microbiology

Multiple Antibiotic Resistance in Pseudomonas aeruginosa: Evidence for Involvement of an Efflux Operon KEITH POOLE,* KATHLEEN KREBES, CATHERINE McNALLY, AND SHADI NESHAT Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada Received 7 June 1993/Accepted 2 September 1993

An outer membrane protein of 50 kDa (OprK) was overproduced in a siderophore-deficient mutant of Pseudomonas aeruginosa capable of growth on iron-deficient minimal medium containing 2,2'-dipyridyl (0.5 mM). The expression of OprK in the mutant (strain K385) was associated with enhanced resistance to a number of antimicrobial agents, including ciprofloxacin, nalidixic acid, tetracycline, chloramphenicol, and streptonigrin. OprK was inducible in the parent strain by growth under severe iron limitation, as provided, for example, by the addition of dipyridyl or ZnSO4 to the growth medium. The gene encoding OprK (previously identified as ORFC) forms part of an operon composed of three genes (ORFABC) implicated in the secretion of the siderophore pyoverdine. Mutants defective in ORFA, ORFB, or ORFC exhibited enhanced susceptibility to tetracycline, chloramphenicol, ciprofloxacin, streptonigrin, and dipyridyl, consistent with a role for the ORFABC operon in multiple antibiotic resistance in P. aeruginosa. Sequence analysis of ORFC (oprK) revealed that its product is homologous to a class of outer membrane proteins involved in export. Similarly, the products of ORFA and ORFB exhibit homology to previously described bacterial export proteins located in the cytoplasmic membrane. These data suggest that ORFA-ORFB-oprK (ORFC)-dependent drug eftlux contributes to multiple antibiotic resistance in P. aeruginosa. We propose, therefore, the designation mexAB (multiple efflux) for ORFAB.

iron-deficient succinate minimal medium (see below) containing 0.5 mM 2,2'-dipyridyl. Growth media. Iron-deficient succinate minimal medium has been described elsewhere (60) and was made iron sufficient by the addition of FeSO4 (100 ,uM). For culturing of E. coli cells, glucose (0.4% [wt/vol]) replaced succinate in the abovedescribed medium. In some experiments, a phosphate-sufficient N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered minimal medium (28) was used as the iron-deficient medium and was similarly made iron sufficient by the addition of 100 ,uM FeSO4. Amino acids (1 mM) and thiamine-HCl (30 ,uM) were added to growth media as required. L broth (16) was used as the rich medium throughout. Ampicillin (100 ,ug/ml), carbenicillin (200 ,ug/ml), tetracycline (P. aeruginosa, 100 ,ug/ml; E. coli, 10 ,ug/ml), kanamycin (50 ,ug/ml), and HgCl2 (15 ,ug/ml) were included in growth media as necessary. Solid media were obtained by the addition of Bacto Agar (Difco; 1.5% [wt/vol]). Membrane isolation and SDS-polyacrylamide gel electrophoresis. Outer membranes were prepared by differential Triton X-100 solubilization of isolated cell envelopes (66) or by sucrose gradient density centrifugation (29). Inner membranes were prepared on sucrose gradients (29). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was carried out as described previously (46) with 9 or 14% (wt/vol) acrylamide in the running gel. Purification of OprK. Outer membranes prepared from 8 liters of P. aeruginosa K385 grown overnight in iron-sufficient succinate minimal medium were resuspended in 80 ml of 2% (vol/vol) Triton X-100-20 mM Tris-HCl (pH 8.0)-i M NaCl and centrifuged (180,000 x g; 60 min). The pellets obtained were subsequently reextracted with 80 ml of the same solution and then resuspended in 40 ml of 1% (wt/vol) Zwittergent 3-14 (Calbiochem)-20 mM Tris-HCl (pH 8.0). Following centrifugation (180,000 x g; 60 min), the resultant supernatant containing Zwittergent 3-14-soluble material was recovered and loaded onto a 5-ml DEAE-Sepharose CL-4B column (1.5

Pseudomonas aeruginosa is a clinically significant pathogen characterized by intrinsic resistance to a number of antimicrobial agents. Moreover, problems with the development of resistance to agents generally exhibiting potent antibacterial activity against this organism (e.g., carbepenems and fluoroquinolones) are encountered with increasing frequency (12, 14, 19, 41, 43, 47, 48, 54, 55, 65). In addition, cross-resistance to chemically unrelated antibiotics can be associated with fluoroquinolone resistance (33, 43, 48, 54, 55, 62, 65). In vitro studies of fluoroquinolone-resistant strains exhibiting cross-resistance have indicated that resistance is attributable to decreased drug accumulation resulting from alterations in outer membrane permeability (12, 14, 19, 24, 41, 43, 47, 65). In some instances, this conclusion stems from the identification of novel outer membrane proteins in these mutants (24, 33, 43, 47). Fluoroquinolone resistance has also been reported for Escherichia coli, for which an energy-dependent efflux mechanism has been implicated (10, 11, 38). Interestingly, cross-resistance to unrelated antibiotics is also a property of some of these mutants (32, 37, 38), and multiple-antibiotic-resistant E. coli also shows cross-resistance to fluoroquinolones (11). Recently, we identified an operon (ORFABC) in P. aeruginosa apparently involved in pyoverdine secretion (57). We report here the characterization of ORFC, which encodes an outer membrane protein whose overproduction is associated with multiple antibiotic resistance. Moreover, we demonstrate that the products of ORFABC show substantial homology to bacterial efflux proteins. MATERIALS AND METHODS Strains and plasmids. The bacterial strains and plasmids used in this study are described in Table 1. Strain K385 is a K372 derivative isolated as a spontaneous mutant growing on * Corresponding author. 7363

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7364

TABLE 1. Bacterial strains and plasmids Strain or plasmid

Strains P. aeruginosa PA06609 K372

Description'

K635

met-9011 amiE200 rpsL pvd-9 Derivative of PA06609 deficient in production of pyochelin and the ferripyochelin receptor Dipyridyl-resistant derivative of K372 overproducing an outer membrane protein of 50 kDa and inner membrane proteins of 40 and ca. 110 kDa K372 ORFA::tet K372 ORFC::QlHg ilv-220 thr-9001 leu-9001 met9011 pur-67 aphA Pyoverdine-deficient derivative of

K636

ML5087 K635 (ORFB::mini-Tn]O-kan)

K385

K590 K613 ML5087

E. coli 5K K38 S17-1

Source or

reference

thr lacZ rpsL thi ser hsdR hsdM X+ (HfrC) relA1 thi pro hsdR recA Tra+

Plasmids

pADD214 pPV1

Mini-D replicon derived from phage D3112; used for in vivo cloning in P. aeruginosa; Tcr pADD214 derivative carrying P. aeruginosa chromosomal DNA which restores growth of P.

aeruginosa K437 on dipyridylcontaining iron-deficient pAK1900 pGP1-2

pT7-5

pT7-6

pSUP301

medium E. coli-P. aeruginosa shuttle cloning vector; Apr Cbr pACYC177 derivative carrying the phage T7 RNA polymerase gene under X P,. control and the c1857 repressor gene; Kmr pBR322 derivative carrying a multicloning site downstream of the strong gene 10 promoter of phage T7; Apr Same as pT7-5 but with the multicloning site in the

opposite orientation; Apr pACYC177 derivative carrying the Mob (mobilization) site of plasmid RP4; Apr Kmr

" Apr, ampicillin resistance; Cb', carbenicillin resistance; Tcr, tetracycline resistance; Kmr, kanamycin resistance.

by 3.0 cm; Pharmacia-LKB) equilibrated with 1% Zwittergent 3-14-20 mM Tris-HCl (pH 8.0) (column buf-er A). The column was subsequently washed with 10 ml of column buffer A and then with 5 ml of column buffer A containing 0.1 M NaCl. Bound protein was eluted from the column with a 40-ml linear gradient of NaCl (0.1 to 0.4 M) in column buffer A and collected in 1-ml fractions. Fractions enriched for OprK (as determined by SDS-polyacrylamide gel electrophoresis) were pooled and dialyzed overnight against 1% Zwittergent 3-14-10 mM Na2HPO4-NaH2PO4 buffer (pH 7.0)-0.1 M NaCl (column buffer B). The dialyzed sample was then applied to a 3-ml

hydroxyl apatite column (1 by 4 cm; Bio-Rad Laboratories, Mississauga, Ontario, Canada) equilibrated with column buffer B. After the column was washed with 10 ml of column buffer B, bound protein was eluted with a 40-ml linear gradient of Na2HPO4-NaH2PO4 buffer (pH 7.0) (10 to 350 mM) in 1% Zwittergent 3-14-0.1 M NaCl. One-milliliter fractions were collected, and those containing pure OprK (as determined by SDS-polyacrylamide gel electrophoresis) were pooled and stored at - 20°C. CNBr digestion of OprK and isolation of digestion products. CNBr digestion of OprK was based on the methodology described by Schultz and Oroszlan (67). In brief, purified OprK (500 pLg) was incubated with CNBr (4 mg/ml in 70% formic acid) at 37°C for 24 h in a final volume of 500 pL.. Following dilution with I ml of 3-mercaptoethanol (0.2%), the mixture was lyophilized and resuspended in 100 pl. of water. Lyophilization was repeated twice more, and OprK was finally resuspended in 20 .1l of SDS-polyacrylamide gel electrophoresis sample buffer (46) and electrophoresed on 14% (wt/vol) polyacrylamide gels. Electrophoresed proteins were subsequently transferred electrophoretically to ProBlott membranes (Applied Biosciences Inc., Mississauga, Ontario, Canada) in

3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer as detailed by the manufacturer. Proteins were visualized with Coomassie blue according to a procedure described by the manufacturer, and bands corresponding to digestion products of OprK were excised with a razor blade. An N-terminal amino acid sequence determination was carried out on electroblotted material at the Centres of Excellence Core Facility for Protein and DNA Chemistry at Queen's University. Antimicrobial susceptibility testing. The susceptibilities of P. aeruginosa strains to a number of antimicrobial agents were tested by inoculating 1-ml cultures of iron-deficient BM2 succinate minimal medium containing serial twofold dilutions of each antimicrobial agent with 5 x 106 organisms. Growth was assessed visually after 18 h of incubation at 37°C. The MIC was defined as the lowest concentration of antimicrobial agent that inhibited visible growth. DNA methodology. Plasmid DNA was routinely prepared by the alkaline lysis procedure (64). Restriction endonucleases and T4 DNA ligase were obtained from Gibco-BRL or Pharmacia-LKB and used according to manufacturer's instructions or as described by Sambrook et al. (64). Transformation of E. coli (64) and P. aeruginosa (4) with plasmid DNA has been described elsewhere. Restriction fragments were isolated, as required, from agarose gels (0.8 to 1.5% [wt/vol]) with Geneclean (Bio 101, Inc., La Jolla, Calif.) or Prep-a-gene (Bio-Rad) glass matrices as detailed by the manufacturer. Subcloning of DNA was performed initially with E. coli 5K prior to its introduction into P. aeruginosa. Cloning and sequencing of ORFC. ORFC was cloned together with ORFA and ORFB (57) by use of the in vivo cloning system described by Darzins and Casadaban (15). Plasmid DNA carrying ORFC was prepared on CsCl2 gradients (64) and sequenced at the Centres of Excellence Core Facility for Protein and DNA Chemistry at Queen's University. An overlapping sequence from both strands was obtained by use of a series of custom-synthesized primers. Nucleotide and deduced amino acid sequences were analyzed with the PC Gene software package (Intelligenetics Inc., Mountain View,

Calif.). Expression of cloned genes in pT7 vectors. To identify the products of cloned genes, the phage T7-based expression system of Tabor and Richardson (70) was used. In brief, E. coli K38 harboring T7 RNA polymerase plasmid pGPI-2 and a recombinant pT7 plasmid carrying cloned genes of interest was

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MULTIPLE ANTIBIOTIC RESISTANCE IN P. AERUGINOSA

grown to the log phase in L broth containing the appropriate antibiotics. Cells (200 pll) were harvested by centrifugation and washed twice in an iron-replete glucose minimal medium containing thiamine before being resuspended in 1 ml of this medium supplemented with 1 mM concentrations of all amino acids, except for methionine and cysteine. Following incubation at 37°C for 30 to 45 min, the cultures were shifted to 42°C for 15 min, at which time rifampin (400 p.g/ml) was added. After a further 15 min of incubation at 42°C, the cells were pulse-labeled with Tran35Slabel (20 VLCi; ICN Biomedicals Canada Ltd., Mississauga, Ontario) for 5 min at 37°C. The cells were then harvested by centrifugation, resuspended in 100 [lI of gel loading buffer (46), and heated at 95°C for 5 min. The resulting whole-cell extracts were resolved on SDS-polyacrylamide gels, which were subsequently stained and destained briefly, dried, and exposed to Cronex-4 X-ray film (Dupont Canada, Mississauga, Ontario) for 24 h. In vitro mutagenesis and gene replacement. To construct P. aeruginosa strains mutated in ORFC (see Fig. 6), ORFC was first isolated on a 4-kb PstI fragment derived from pPV20 and then cloned into the unique PstI site on pSUP301 (68) to yield pPV21. Plasmid pPV21 was partially digested with BamHI, which cuts once in pSUP301 and once in ORFC, and fulllength DNA was recovered from agarose gels. Full-length DNA was identified by simultaneously running an EcoRI digest of the same DNA. The mercury resistance determinant of plasmid pPH45fQHg (22) was isolated as a BamHI fragment which was subsequently ligated to BamHI-digested (partially) pPV21 and transformed into E. coli 5K. Plasmids carrying the mercury resistance determinant within ORFC were identified via restriction analysis. One such plasmid (pPV22) was transformed into mobilizing E. coli S17-1 (68), which was subsequently conjugated with P. aeruginosa strains by pelleting equal volumes (100 [I1) of overnight cultures of donor (grown at 37°C) and recipient (grown at 42°C) cells in microcentrifuge tubes, resuspending the cells in 25 [L1 of L broth, and spotting the cells onto the center of L broth plates. After overnight incubation at 37°C, bacteria on the plates were suspended in 1 ml of L broth and subsequently plated on HgCl2-containing L broth plates. P. aeruginosa recipients carrying chromosomal ORFC::flHg were HgCl2 resistant and carbenicillin sensitive.

RESULTS Identification of a 50-kDa outer membrane protein in mutants of P. aeruginosa growing in the presence of 2,2'dipyridyl. In an attempt to identify outer membrane components involved in iron acquisition in P. aeruginosa, spontaneous mutants of strain K372 able to grow in the presence of growth-inhibitory concentrations of the nonmetabolizable iron chelator 2,2'-dipyridyl (0.5 mM) were isolated, and outer membranes were screened for the presence of novel proteins. It was reasoned that the ability to grow under such ironrestricted conditions would necessitate an improvement in the iron-acquiring ability of the mutants and that their characterization might identify outer membrane proteins involved in iron uptake. A number of mutants capable of growth in the presence of 0.5 mM 2,2'-dipyridyl were recovered, and many of these (e.g., strain K385) expressed high levels of a 50-kDa outer membrane protein (Fig. IA, compare lanes 1 and 2) which we designated OprK by using the nomenclature suggested by Hancock et al. (30). OprK was not repressible by iron in K385 (Fig. IA, lane 4), even at 200 p.M FeSO4 (data not shown), and was not inducible in the parent strain during growth in phosphate-buffered (BM2) iron-deficient succinate minimal medium (Fig. IA, compare lanes I and 3). Induction

B

A v

7365

E

-4

12 34 FIG. 1. Cell envelope proteins of P. aeruginosa K372 and its OprK-overproducing derivative K385. (A) Outer membrane proteins of K372 (lanes I and 3) and K385 (lanes 2 and 4) grown in iron-deficient (lanes 1 and 2) and iron-sufficient (lanes 3 and 4) BM2 succinate minimal medium. OprK is indicated by an arrowhead. (B) Cytoplasmic membrane proteins of K385 (lane 1) and K372 (lane 2) grown in iron-deficient succinate minimal medium. Proteins overproduced in K385 are indicated by arrowheads. Molecular mass standards (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 42.7 kDa; carbonic anhydrase, 31 kDa; and soybean trypsin inhibitor, 21.5 kDa) are indicated by horizontal lines (from top to bottom).

of the protein was, however, achieved by growth in HEPESbased iron-deficient minimal medium (Fig. 2, lane 5). In addition, extended (24 to 36 h) incubation of K372 in the presence of 0.5 mM 2,2'-dipyridyl resulted in the growth of K372 and the concomitant induction of OprK (Fig. 2, lane 3). Interestingly, maximal production of the protein was achieved by growth in the presence of ZnSO4 (Fig. 2, lane 7). Susceptibility of K385 to antimicrobial agents. The production of a 50-kDa outer membrane protein was previously observed for mutants of P. aeruginosa resistant to streptonigrin (56), and K385 exhibited enhanced resistance to this compound (Table 2). This association between the production of a 50-kDa outer membrane protein and resistance to an antimicrobial agent was intriguing, since a number of previously reported quinolone-resistant P. aeruginosa strains also produced novel outer membrane proteins with a ca. 50-kDa molecular mass (24, 33, 43). Furthermore, these mutants showed cross-resistance to additional nonquinolone antibiotics. Examination of the antibiotic susceptibility of K385 revealed that it too exhibited resistance to quinolones (ciprofloxacin and nalidixic acid) as well as to chloramphenicol and tetracycline (Table 2). Overproduction of cytoplasmic membrane proteins in K385. Quinolone-resistant E. coli and multiple-antibiotic-resistant E. coli have been described (11, 32, 37, 38), and resistance has, in part, been attributable to an efflux component located in the cytoplasmic membrane (10). To determine whether the resistance phenotype of K385 was characterized by alterations in the cytoplasmic membrane, in addition to overproduction of outer membrane protein OprK, cytoplasmic membrane fractions were prepared from K385 and its parent and examined

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J. BAC-FERIOL.

4 5

6 7 8

FIG. 2. Induction of OprK in P. aeruginosa K372. Outer membranes were prepared from P. aeruginosa K372 cultured in irondeficient BM2 succinate minimal medium (lane 2), iron-deficient BM2 succinate minimal medium with dipyridyl (0.5 mM) (lane 3), ironsufficient HEPES-buffered minimal medium (lane 4), iron-deficient HEPES-buffered minimal medium (lane 5), iron-deficient BM2 succinate minimal medium (lane 6), and iron-deficient BM2 succinate minimal medium with ZnSO4 (0.1 mM) (lane 7). Lanes 1 and 8 show outer membranes of K385 grown in iron-deficient BM2 succinate minimal medium. OprK is indicated by arrowheads.

SDS-polyacrylamide gels. A protein of ca. 40 kDa, absent in K372, was readily observed in the cytoplasmic membrane preparations of K385 (Fig. 1B, lane 1). In addition, there appeared to be increased staining in the region of a diffuse band at ca. 108 kDa in the cytoplasmic membrane preparations of K385 (Fig. IB, lane 1), consistent with increased production of this protein or production of an additional protein of a similar size. Interestingly, we recently identified an operon of at least three genes (ORFABC) (57) (Fig. 3), the first two of which encode products of 40 and ca. 108 kDa and predicted to occur in the cytoplasmic membrane (57). Moreover, mutants defective in these genes show a decrease ability to grow in the presence of 2,2'-dipyridyl (57). Given that the ORFAB genes are important for growth on dipyridyl and encode products of the same molecular masses as the cytoplasmic membrane proteins identified in a mutant (K385) growing on elevated levels of dipyridyl suggest that these cytoplasmic membrane proteins are the products of ORFAB. Moreover, they suggest that OprK is the product of the third gene of this operon, on

ORFC. Nucleotide

of ORFC. A portion of ORFC was previously sequenced (57); to determine whether OprK was the product of ORFC, its sequence was completed (Fig. 4). ORFC, which begins 2 bp downstream from the stop codon of ORFB, encompasses 1,430 bp and is predicted to encode a product of 476 amino acids (excluding the initiation methionine) and with a molecular mass of 51,481 Da. The deduced ORFC product possesses a sequence at its N terminus characteristic of signal sequences as well as a putative lipoprotein signal peptidase cleavage site (75). The predicted mature sequence

polypeptide comprises 460 amino acids and has a molecular mass of 50,010 Da. The expression of ORFC on a 6.5-kb SmaI-HindIll fragment (Fig. 3B) in E. coli by use of the phage T7-based expression system (70) yielded a major product of 49 kDa and a minor product of 51 kDa (Fig. 5). This result was in excellent agreement with the predicted molecular masses of the mature and precursor forms, respectively, of the ORFC product and with the estimated molecular mass of OprK. A weakly detected band running above the 97-kDa molecular mass marker is apparently derived from ORFB, most of which is present on the 6.5-kb Smal-HindlIl fragment. N-terminal amino acid sequencing of OprK was unsuccessful, as might be expected for a possible lipoprotein. Problems were also encountered with CNBr digestion of the protein, owing to the apparent acid lability of the protein, although a 43-kDa digestion product was recovered in sufficient quantity for N-terminal amino acid sequencing. The N-terminal sequence obtained for this degradation product (ALXNNRXL) matches the predicted ORFC amino acid sequence beginning at residue 73 (bp 228). Moreover, an ORFC-derived product beginning here would have a molecular mass of 44,110 Da, in good agreement with the estimated size of the OprK-derived CNBr digestion product which was sequenced. Thus, OprK is, indeed, the product of ORFC, which should now be referred to as oprK. Furthermore, it is likely that the cytoplasmic membrane proteins identified in K385 are the products of ORFAB. Characterization of mutants defective in ORFABC. As the mutation in strain K385 has yet to be mapped or fully characterized, it is possible that additional alterations which may be responsible for the multiple antibiotic resistance phenotype of strain K385 occur. To demonstrate a direct involvement of ORFABC in multiple antibiotic resistance, then, mutants specifically deficient in the ORFABC products were constructed and examined for antibiotic susceptibility. An ORFA mutant had been constructed previously by insertion of the tet gene of plasmid pBR322 into the cloned gene, which was then introduced into P. aeruginosa by gene replacement (57). Similarly, a stable ORFB mutant had been obtained earlier by insertion of a mini-Tn]O-kan element into the cloned gene, which was also introduced into P. aeruginosa by gene replacement (57). Finally, an ORFC mutant was constructed (Fig. 6) by insertion of the HgCl2 resistance determinant of pHP45flHg (22) into the cloned gene, which was then introduced into P. aeruginosa by gene replacement. Interestingly, attempts at introducing these mutations into wild-type P. aeruginosa PAO1 or mutant strain K385 were unsuccessful, despite repeated attempts. This was unfortunate, since it precluded direct confirmation that the ORFAB genes were responsible for the production of the cytoplasmic membrane proteins overproduced in K385. Moreover, while mutated ORFA and ORFB were readily introduced into strain K372, the recovery of K372 carrying the ORFB::mini-TnlO-kan mu-

TABLE 2. Susceptibilities of P. aeruginosa strains to selected antimicrobial agents MIC' of: Strain

K372 K385

DIP

SN

CIP

NAL

CAM

TET

0.5 1.0

10 40

0.125 1.5

62.5 250

12.5 100

2.5 25

a Values are reported in micrograms per milliliter, except that values for dipyridyl are reported as micromolar concentrations. DIP, 2,2'-dipyridyl; SN, streptonigrin; CIP, ciprofloxacin; NAL, nalidixic acid; CAM, chloramphenicol; TET, tetracycline.

MULTIPLE ANTIBIOTIC RESISTANCE IN P. AERUGINOSA

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1 kb PstI EcoRI PstI SmaI BamHI HindIII PstI 1+ jP" PstI SstI

il

PstI

SphI SphI

BamHI XhoI

I

II

A

A-

r

w r

v

Fv

ORFB

ORFA

ORFC

att

(A) HindIII

HindIII

(B) SmaI pT7-5 >>

HindIII