A sulfone group-labeled TEM-DNA probe: comparison with a 32P-labeled probe in dot-hybridization

Journal of Biochemical and Biophysical Methods, 16 (1988) 301-310 Elsevier

301

BBM 00678

A sulfone group-labeled TEM-DNA probe: comparison with a 32p-labeled probe in dot-hybridization M. Jouvenot. F. Descotes, A. Remy-Martin and G.L. Adessi I N S E R M U 198, 240 route de Dole, 25000 Besan~on, France

(Received 29 March 1988) (Accepted 7 April 1988)

Summa~ A non-radioactive DNA probe for the TEM-type ]Mactamase gene was obtained by using the 'Chemiprobe' system. It was used along with a 32p-labeled TEM probe to screen for TEM fl-lactamase gene in 107 bacterial isolates representing 7 Gram-negative genera and previously classified as TEM-posirive or negative. The DNA to be tested was extracted from these bacterial isolates by the Bimhoim-Doly method and, after blotting into charged nylon membranes, it was submitted to hybridization with either the TEM 'Chemiprobe' or the 32p-TEM probe. The TEM 'Chemiprobe' could detect as few as 25 pg specific DNA if it was used at a concentration of 5 ng per cm2 of membrane. The results obtained by both probes were concordant in 93.5% of the entire sample. The TEM 'Chemiprobe' was specific since only one false positive was observed. Furthermore, it appeared at least as sensitive as the 32p-labeled TEM probe. As the dot-hybridization with the sulfone-labeled probe was sensitive, simple and easy to perform, it will be useful for large-scale screening in clinical laboratory. Key words: Gram-negative bacteria; TEM-type /3-1actamase gene; Dot-hybridization; Sulfone-labeled DNA probe

Introduction The study of genes encoding antibiotic resistance has benefitted from the use of nucleic acid hybridization methods [1]. Among the fl-lactamase genes, the TEM gene has been the most widely studied by using the DNA probe technology [2-5]. Most of the probes described are labeled with 32p which is detected with, high specificity and sensitivity by autoradiography. However, these radioactive probes Correspondence address: M. Jouvenot, INSERM U 198, 240 route de Dole, 25000 Besan~on, France. 0165-022X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

302 have limited usefulness in the chemical laboratory because of their short half-life and inherent health risks. As an alternative, enzymatic detection methods have recently been developed [6-101. They overcome many of the problems associated with isotopic methods and efforts are continuously being made in order to provide a greater sensitivity. Biotin and its derivatives are the most frequently used non-radioactive markers of nucleic acid probes [6,7]. More recently, ilrmmnochemically detectable haptens have been developed as Mternatives to biotin. Thus, guanine residues in DNA (or RNA) can be modified using N-acetoxy-N-2-acetyl-aminofluorene (AAF) or its 7-iodo-derivative; antibodies produced against guanosine-acetylaminofluorene are then used to identify the hybridized probe [10,11]. Also, other haptens, such as sutfone groups can be introduced into DNAs [12,13]. With respect to fl-lactamase genes, biotinylated probes are the only non-isotopic probes which have been evaluated. It appears that they can be used successfully when the DNA is purified (e.g., through the Southern blot method), but false positive reactions occur with DNAs extracted by the Birnboim-Doly method and with crude bacterial preparations (e.g., boiled lysates and colony blots) [14]. In our laboratory, the development of a TEM B-tactamase probe allowed a prior study in which isoelectric focusing (IEF) was used together with colony hybridization using 32p to monitor classes of fi-lactamases in 328 isolates of gram-negative bacteria [3]. In the present study, we evaluated the same TEM probe but after labeling by a sulfone group. The detection of TEM-fi-lactamase was assayed on 107 isolates, previously classified as TEM-positive or negative. Two probes, the 32p probe and the sulfonated probe, were tested on extracts obtained using the Birnboim-DoIy method.

Materials and Equipment Bacterial strains

One hundred and seven Gram-negative bacteria were isolated from different patients in the Medical School of Besancon. They belonged to the following seven genera: Escherichia (49 strains), Klebsiella (25 strains), Serratia (13 strains), Providencia (10 strains), Enterobacter (5 strains), Pseudomonas (3 strains) and Citrobacter (2 strains). They were classified as TEM (TEM-1 or TEM-2) positive or negative, by isoelectric focusing and by testing with a 32p-labeled TEM DNA probe in colony blotting [3]. The reference bacterial strains (HB 101) were provided by P. Courvalin (Paris, France), strain HB 101 (pBR322) by Pi Bennett (Bristol, U.K.) and strain PU 21 (RPL11) by G. Jacoby (Boston, MA). Materials and reagents

The Zeta Probe membranes were obtained from Bio-Rad laboratories, Richmond, CA and the [a-3zp]dCTP was acquired from New England Nuclear, Boston, MA. Bethesda Research Laboratories, Gaithersburg, MD, provided the nick-translation

303 kit and the Hybri-slot T M Manifold, whilst the chemiprobe kit was supplied b y Orgenics Ltd. Yavne, Isra61. The X-ray films ( X - O M A T RP) were obtained from K o d a k and Lightning-Plus (Du Pont Cronex; Du Pont Co., Wilmington, DE), intensifying screens were acquired from Euromedica, France. The controlled environment incubator shaker was obtained from N e w Brunswick Scientific Co. (Edison, N J, U.S.A.). 1 × T A E buffer ( p H 8.0) was 0.04 M Tris, 0.04 M sodium acetate and 0.002 M EDTA. 1 x SSPE (pH 7.4) consisted of 0.15 M NaC1, 10 m M N a H z P O 4 and 1 m M EDTA. 1% B L O T T O was composed of 1% non-fat powdered milk (Gloria) in sterile deionized water supplemented with 0.02% sodium azide [151. 1 x SSC (saline citrate solution) consisted of 0.15 M NaC1 and 0.015 M sodium citrate. The prehybridization solution no. 1 consisted of 50% deionized formamide, 4 × SSPE, 1% sodium dodecyl sulfate (SDS), 0.5% B L O T T O and 0.5 mg of denatured salmon sperm D N A per ml. The hybridization solution no. 1 was the same as the prehybridization solution no. 1 but without the salmon DNA. The prehybridization solution no. 2 was made up of 50% deionized formamide. 1 M NaC1, 1% SDS, 10% dextran sulfate and 0.25 mg of denatured salmon sperm DNA. The hybridization solution no. 2 was the same as the prehybridization solution no. 2 but without the salmon DNA. The chromogenic substrate system was composed of 0.30 mg of nitro-blue tetrazolium and 0.20 mg of 5 bromo-4-chloro-3-indolyl phosphate per ml of buffer TNM. T N M buffer was 100 m M Tris-HC1 (pH 9.5), 100 m M NaC1 and 5 m M MgC12.

Methodology Preparation of DNA spots The plasmid D N A was extracted from the bacterial strains by the alkaline method of Birnboim and Doly [16]. After the final ethanol precipitation, the extracted D N A s were diluted in sterile distilled water. They were quantitated spectrophotometrically by measuring absorbance at 260 nm. Then they were boiled for 5 rain and chilled rapidly in an ice-water bath. These denatured D N A samples were spotted, by means of the Hybri-slot T M Manifold, onto nylon membranes presoaked in 4 × TAE. The membranes were air dried, then baked for 2 h at 80 o C in vacuo, and finally stored in a dry place until used.

Preparation and labeling of the TEM gene probe The host strain for the probe D N A source was E. coli HB 101 (pBR322). After plasmid isolation [17], the sequential digestion with EeoRI and HinfI yielded a I kb D N A fragment which was specific for the T E M gene [2,3]. This T E M probe was used in hybridization studies after appropriate labeling,

304 The radioactive labeling of the T E M probe was achieved with [~-32p]dCTP and a nick-translation kit according to the manufacturer's protocol. The specific activity of the probe ranged between 107 and 10 s cpm per b~g of D N A . The nonisotopic probe was prepared by inserting an antigenic sulfone group into cytosine moieties of the denatured D N A [121 and this reaction was performed with the chemiprobe kit as recommended by the manufacturers. The sulfonated probe was used just after preparation although it was known to be stable for m a n y months.

Hybridization to DNA spots Method with the 32P-labeled probe

The membranes were treated at 48 ° C (in heat-sealed plastic bags) with prehybridization solution no. 1 (100 ~1 per cm a of membrane)° After 6 h of incubation, the prehybridization solution was replaced with the hybridization solution no. 1 containing the denatured 32p-labeled probe (1{] 4 cpm per cm a of membrane), and incubation was continued for 15 h at 4 8 ° C with continuous shaking. Once the hybridization was completed, the membranes were washed four times, each wash lasting 10 min, in a large volume of 0.1% S D S / 2 × SSC at room temperature. They were then washed twice, for 1 h each time, in a large volume of 0.1% S D S / 1 × SSC at 68 ° C. The hybridized membranes were air dried and exposed to X-ray film with an intensifying screen for 20 h at room temperature. Autoradiographs were developed with an automatic X-ray developer. Method with the chemiprobe The membranes were treated at 42 ° C (in heat-sealed plastic bags) with ~rehybridization solution no. 2 (100 ~1 per cm 2 of membrane). After 6 h of incubation, the prehybridization solution was replaced with the hybridization solution no. 2 containing the denatured modified probe (at a concentration ranging between 2.5 n g / c m 2 and 25 n g / c m 2 of membrane), and incubation was continued for 6 - 1 4 h at 4 2 ° C with gentle shaking. After the hybridization, the membranes were washed twice, each wash lasting 5 min, in a large volume of 2 × SSC at room temperature. They were then washed again, for 30 rain. in a large volume of 1% S D S / 2 × SSC~ at 42 ° C. A final washing (30 min) was carried out in a large volume of 0.1 × SSC at room temperature. Visualization of the hybrids was realized by an enzymatic sandwich immune reaction following the procedure suggested b y the manufacturer: after the blocking of the membrane (to prevent non-specific immune reactions), monoclonal anti-modified D N A antibodies were applied (for 1 h at room temperature) and washes removed the excess of antibodies; an alkaline phosphatase anti-immunoglobulin conjugate was then added (for 1 h at room temperature) and, after appropriate washes, the chromogemc substrate system was applied. After incubation ( 1 - 2 h at 37 ° C), the blue-violet color development was terminated by washing the m e m branes in tap water.

Concentration of the chemiprobe tn a first series of experiments, we determined the amount of the chemiprobe to be used. For this purpose, different amounts of plasmidic D N A extracted from HB101 (pBR322), ranging from 20 ng to 0.025 ng, were spotted onto four nylon

305

a

b

c

d

e

f

A 2

Fig. 1. Detection of pBR322 DNA in dot-hybridization assays, using the chemically modified TEM probe. (A) 5 ng of chemiprobe/cm2: (B) 2.5 ng of chemiprobe/cm2. Amounts of pBR322 DNA were as follows: la, 20 ng; lb. 15 ng; lc. 10 ng; ld. 5 ng; le, 2.5 ng; If, 1 ng; 2a. 0.50 ng; 2b, 0.25 ng; 2c, 0.10 ng; 2d, 0.050 ng; 2e. 0.025 ng. The negative control, 2f, consisted of 20 ng of DNA from PU21 (RPLll). membranes, each of which was hybridized with one of the following amotmts of the T E M chemiprobe: 2.5, 5, 10 or 25 n g / c m 2. Fig. 1 shows the results obtained with 5 ng and 2.5 n g / c m 2 of membrane. For each chemiprobe concentration, all D N A spots ranging between 20 ng and 0.5 ng were positive. Amounts of pBR322 D N A lower than 0.5 ng were not detectable with the TEM probe at 2.5 n g / c m 2, but they were with the three other probe concentrations. Furthermore, with a low amount of probe (2.5 ng/cm2), there was a poor correlation between the amount of D N A and the colour intensity. The negative control [DNA from PU21 ( R P L l l ) ] did not hybridize even at a high level of TEM probe. On the basis of these results, we selected the 5 ng per cm2 probe concentration which appeared sensitive enough for our investigations into wild strains. This point will be discussed later. For the 32p probe we used the concentration of 0.1 n g / c m 2 which is generally suitable for a good sensitivity and a low background.

Detection of T E M D N A in wild strains When tested with D N A extracted b y the Birnboim-Doly procedure and blotted onto nylon membranes, both dot-hybridization systems were able to distinguish between known TEM-positive [HB101 (pBR322)] and TEM-negative organisms [PU21 (RPLll)] (Fig. 2). The amount of D N A extracted from the 107 wild strains was sufficient to achieve spots with undiluted preparations (concentration ranging from 1 ng to 20 ng) and diluted preparations (concentration ranging from 0.05 ng to 1 ng). After visualization of the hybrids (Fig. 2), a comparison between both dot-hybridization methods

306

c

k

A

2 Fig. 2. D e t e c t i o n of the T E M D N A in reference and wild strains, using dot-hybridization assays. (A) Use of the T E M c h e m i p r o b e ; (B) use of 32p-TEM probe. A l l spots were D N A s from wild strains except the foUowing: l c a n d 2c, D N A f r o m PU21 ( R P L l l ) ; l k a n d 2k, D N A f r o m HB101 (pBR322). A m o u n t s of D N A were as follows: row 1, r a n g i n g b e t w e e n t n g a n d 20 ng; row 2, r a n g i n g b e t w e e n 0.05 n g a n d 1 ng.

could be made on the basis of the undiluted spots. This is summarized in Table 1. For 100 extracts in groups A and B (93.5%), the presence orabsence of the TEM gene was established by both dot-hybridization methods. For 7 extracts in groups C and D (6.5%), results were discordant: so, for 5 extracts in group C (4.7%), a positive result was observed with the TEM-chemiprobe dot-hybridization only, and for 2 extracts in group D, a positive result was observed with the 32p-probe dot-hybridization only. If the interpretation was made on the basis of the diluted D N A spots (between 0.05 ng and 1 ng), the concordance between both dot hybridization methods fell to 81.3%. Among the D N A sanaples which were positive

TABLE 1 COMPARISON METHODS a

BETWEEN

RESULTS

OBTAINED

BY

BOTH

DOT-HYBRIDIZATION

Group

No. of strains

Dot-hybridization with the TEM chemiprobe

Dot-hybridization with the 32 P - T E M probe

Colony blotting and I E F b

A

53

+

+

+

B

45 2

-

-

+

C

D

1

+

-

-

4

+

-

+

2

--

+

+

a This interpretation was drawn from the D N A dots ranging between 1 n g a n d 20 ng. b I E F , isoelectfic focusing.

307 when tested at a concentration between 1 ng and 20 ng, 79.3% remained positive after a twenty-fold dilution if the chemiprobe was used and 74.5% remained positive after dilution if the 3Zp probe was used. As shown in Table 1, for further interpretation, the results of the present study were compared with the results previously obtained [3] by isoelectric focusing (presence of TEM enzyme) and colony blotting (presence of TEM DNA) methods. So, several points should be emphasized. There were 102 concordant results (95.3%) between isoelectric focusing (IEF)/colony blotting and the chemiprobe dot-hybridization and 101 concordant results (94.3%) between IE F/ col ony blotting and the 3ZP-probe dot-hybridization; the concordance between the four methods available (the two dot-hybridization methods, the colony blotting and the IEF method) was 91.6%; among the hundred concordant results in both dot-hybridization methods, 2% (a part of group B) were not in agreement with those previously obtained; among the five cases in group C (positive with the chemiprobe) four appeared really TEM-positive if IEF and the colony blotting were considered: the two cases in group D (positive with the 32p probe) were TEM-positive in IEF ar~d colony blotting. Discussion

The modification of the cytosine residues is a practical means of preparing nonisotopic hybridization probes. The reagents necessary for the labeling and visualization procedures are commercially available. Compared with other methods for the preparation of nonisotopic probes, the present method is simpler and time saving (particularly, no purification is needed after the probe labeling). Furthermore, it allows the use of nylon membranes that bind nucleic acids very efficiently, are more pliable than nitrocellulose and easier to handle [18]. We chose to test the TEM chemiprobe (comparatively with the 32P-TEM probe) with DNAs extracted by the Birnboim-Doly method for the two following reasons. First, the TEM /3-1actamase gene is plasmidic, so the Birnboim~Doly method is convenient for the isolation of the TEM DNA. Secondly, earlier studies using nonisotopic probes have shown that non-specific reactions arise in colony blotting and, to a lesser extent, with partially purified DNAs (e.g. by the Birnboim-Doly method) [6,14]. According to the manufacturer's recommendations, the chemiprobe must be used at a concentration between 25 ng and 50 ng per cm2 of membrane to obtain optimal sensitivity. Thus, in these conditions, DNA detection may attain sensitivities in the range of picograms. Given that the amount of DNA probe available is often a limiting factor and that the DNA spots to be tested in the present study were higher than 1 ng, we endeavoured to minimize the amount of TEM chemiprobe. Thus, a concentration of 5 ng of TEM chemiprobe per cm2 of membrane appeared sufficient to detect as few as 25 pg specific DNA spotted on nylon membranes. This lowered probe concentration made it possible to obtain conditions closer to those used with the 32p probe (0.1 ng 32p probe/cruZ). The two dot-hybridization methods were equally efficient since their concordance rates with the IEF/colony blotting methods were virtually identical (95.3% for the

308 c h e m i p r o b e d o t - h y b r i d i z a t i o n a n d 94.3% for the 32P-probe d o t - h y b r i d i z a t i o n ) . I n the p r e s e n t study, two strains gave negative results w h a t e v e r the p r o b e u s e d while they were positive in the previous s t u d y ( I E F / c o l o n y blotting) [3], a n d this c o u l d b e d u e to a l a c k of D N A extraction. T h e r e is a high c o n c o r d a n c e b e t w e e n the results o b t a i n e d b y t h e two d o t m e t h o d s (93.5%). A m o n g the d i s c o r d a n t results we c a n d i s t i n g u i s h ( a c c o r d i n g to the p r e v i o u s results in I E F / c o l o n y blotting): one false positive w i t h the T E M c h e m i p r o b e , two false negatives with the T E M c h e m i p r o b e a n d four false negatives w i t h the 3 2 p - T E M p r o b e . T h e false positive result c o u l d b e d u e either to s o m e n o n - s p e c i f i c r e a c t i o n d u r i n g h y b r i d i z a t i o n or visualization of the h y b r i d s or to a w r o n g i n t e r p r e t a t i o n after v~sualization (you c a n m i s t a k e a negative for a slightly positive). A false negative result, in one o r the o t h e r of the two m e t h o d s , c a n b e d u e to a failure to b a k e filters to fix D N A or a failure d u r i n g h y b r i d i z a t i o n itself. H o w e v e r . as a whole, the results show t h a t the c h e m i p r o b e is specific a n d at least as sensitive as the 32p p r o b e in the c o n d i t i o n s used. T h e d e t e c t i o n of T E M D N A b y d o t - h y b r i d i z a t i o n with a c h e m i p r o b e c o m p a r e s favorably with c u r r e n t l y used m e t h o d s a n d m a y h a v e p a r t i c u l a r u t i l i t y in c e r t a i n situations. U n t i l now. n o r h s o t o p l c p r o b e s have g e n e r a l l y p r o v e d u n s u i t a b l e for p a r t i a l l y p u r i f i e d D N A s a n d for colony h y b r i d i z a t i o n [14]. A c c o r d i n g to the p r e s e n t s t u d y p e r f o r m e d with partiaUy p u r i f i e d p r e p a r a t i o n s a n d to e x p e r i m e n t s c a r r i e d o u t in o t h e r l a b o r a t o r i e s [19,20] the c h e m i p r o b e could r e p r e s e n t a s u i t a b l e alternative.

Simplified description of

the m e t h o d and its applications

The objective of the present study was to determine whether a sulfonated DNA probe could be substituted for a 32p-labeled DNA probe to detect the presence of TEM-type/3-1actamase gene in DNA preparations from wild bacterial strains. The DNAs were extracted from E. coli HB101 (pBR322) and from Gram-negative bacteria by the Bimboim-Doly method. They were spotted onto nylon membranes and the DNA sequence for the TEM-type/3-1actamase was detected by using a specific DNA probe. This probe was labeled either with 3Zp by hick-translation or with a sulfo•e group by using the Orgenics chemiprobe kit. The hybrids were detected by autoradiography or by an enzymatic sandwich immune reaction. The hybridizations of the spots of pBR322 (bearing the TEM gene) allowed the determination of the suitable sulfonated probe concentration, i.e. 5 ng per cmz of membrane. Used at this concentration the sulfonated probe appeared as specific and sensitive as the 32p probe (t3.1 ng/cm 2). The sulfonated TEM probe appears suitable for epidemiology of bacterial resistances in dot-hybridization, More generally, the experiments so far performed show that a sulfonated DNA probe has the qualities required for clinical research: without health risks, cost and time effective, with a low non-specific background and sensitive (to a lesser extem than 3217 probes in some conditions such. as single copy gene demonstration).

AeknoMedgments W e t h a n k the investigators w h o p r o v i d e d us w i t h the reference strains. W e t h a n k the Orgenics L t d Society for their generous gift of the first c h e m i p r o b e kit t h a t we used. This w o r k was s u p p o r t e d b y a C N A M T S grant.

309

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