Zbl. Bakt. 277, 22-27 (1992) © Gustav Fischer Verlag, StuttgartlNew York

Identification of Aerobactin Genes in Clinical Isolates of E. coli Using a Non-radioactive DNA Probe YVONNE GRASER and GABRIELE SCHONIAN Institut fur Medizinische Mikrobiologie des Bereiches Medizin (Charite) der HumboldtUniversitat, 0-1040 Berlin, Germany

With 1 Figure' Received July 18, 1991 . Revision received November 25, 1991 . Accepted December 20, 1991

Summary A digoxigenin-Iabelled gene probe was used for the identification of aerobactin genes in 21 E. coli strains from clinical isolates by means of a colony hybridization test. The results were compared with those obtained by previous hybridization experiments with radiolabelled DNA probes as well as with a crossfeeding-assay. It could be demonstrated that only after an additional proteinase K treatment of the filters before hybridization and after a repeated blocking during the immunological detection procedure, specific hybridization signals could be observed.

Zusammenfassung Fur den Nachweis der Aerobactingene bei 21 E. coli-Stammen aus klinischen Isolaten in der Koloniehybridisierung wurde eine Digoxigenin-markierte Gensonde erprobt. Zum Vergleich wurden die Ergebnisse friiherer Hybridisierungen mit radioaktiv markierten DNASonden sowie des biologischen Nachweises der Aerobactinproduktion mit dem Crossfeeding-Test herangezogen. Es konnte gezeigt werden, daR nur nach einer zusatzlichen Proteinase-K-Behandlung der Koloniefilter vor der Hybridisierung und wiederholter Blockierreaktion wahrend des immunologischen Nachweises der Hybride spezifischeHybridisierungssignale beobachtet wurden.

Introduction

E. coli has emerged as an important nosocomial pathogen responsible for a significant proportion of hospital-acquired infections such as urinary tract diseases, septicemia and meningitis. Pathogenic E. coli strains are often found to be associated with specific virulence factors enabling the bacteria to survive and multiply in the host. It has been demonstrated, that the ability to express a high-affinity system of iron uptake,

Identification of Aerobact in Genes in Clinical Isolates of E. coli

23

e.g. aerobactin, correlated with the viru lence for a va riety of E. coli strains isolated from extraintestinal infecti on s (6, 16). The aerobactin system of E. coli was cloned and radiolabelled DNA probes were developed to detect the aerobactin operon by DNA-DNA hybridization (1,2,6,10,13, 14, 15). However, the disadvantages of radiolabelled DNA, i.e, health hazards and short half-life, make the use of DNA hybridization difficult in routine diagnostics and in laboratories with limited equipment. The aim of this work was to develop a non -isotopic gene probe for the detection of the aero bactin system by means of colony hybridization. For this purpose it was necessary to optimize th e test conditions to o btain specific hybridization signals.

Materials and Methods 1. Bacteria. The characteri stics of laboratory strains used in this study are shown in Table 1. The clinical E. coli strain s were isolated from extraintestinal infections and were obtained from the collection of our institute and of the Institute of Infectious Diseases of Children, Berlin-Buch. 2. Detection ofaerobactin production. Aerobacrin production was detected by crossfeeding with E. coli LG 1522 as indicator (5). 3. Probe preparation . The isolation of the plasmid DNA of pABNl was performed by the alkaline lysis method followed by a centrifugation in a cesium-chloride-ethidium bromide density gradient. The 3.4 kb HindIII/BamHl fragment of pABNl DNA was purified from a low-melting-point agaro se gel (0.7%) and labelled with digoxigenin in a random priming reaction using a non-radioactive gene probe labelling and detection kit, provided by Boehringer GmbH (Mannheim, Catalo gue number 1093657) according to the enclosed instructio ns.

Table 1. E. coli K12 strain s and plasmids (Iu = Iron uptake ability, CS = Cloacin sensivity (intact membrane receptor ), Crn" = resistance to chloramphenicol, Apr = resistance to ampicillin) Relevant phenotype conferred by plasmid

Source

Strain

Genorype

HB 101

hsh S, rec A, ara, pro A, lac Y, leu, gal K, rps L, xyl, sup E, mrl

Boye r

LG 1522

ara, azi, fep A, lac, leu, mel, pro C, rps L, tsx, ton A, thi (pCoIV- K30 iuc)

Williams (17)

AN 193

leu, pro , thi, trp, end A. ton A. (piE 745)

lu+,

end A, rhi, pro, hsd R (pABN 1)

lu+, c , Apr (16,3 kb Hind III fragment of pColV-K30 cloned in a pBR 322 derivative)

294

(3)

o , CoIV, Crn", Tcr

Buchholz (4)

Neilands (1)

24

Y. Graser and G. Schonian

4. Colony hybridization . Single colonies of various E. coli strains were cultivated on blotting membranes overlaying Luria-Bertani agar. The fixation of the bacterial DNA to the filters was carried out accord ing to the method of Maniatis (8). Prehybridizat ion, hybridization and detection of the labelled hybrids were performed as recommended by the supplier of the gene probe labelling kit. Hybrid ization was conducted at 68 °C for 3 hour s. The modifications of this method are stated in the text .

Resu lts an d Disc ussion In thi s study, a digoxi genin-labelled aerobactin gene probe was applied for testing 21 clinical E. co li strains obtained from extraintestinal infections (T able 2). For 12 of these strains, the presence of the aerobactin iron uptake system was confirmed by the cro ssfeeding-bioassay and by an previously described hybridization assay using radiolabelled oligo - and polynucleotide probes (11). The remaining 9 strains lacked th e aer ob actin genes (Tab le 2, Fig. 1A).

Tabl e 2. Aerobactin production of 21 E. coli strains from different clinical isolates (B: blood cultu re, L: CSF, W: wound infection, F: fecal isolate; OP: oligonucleotide probe , PP: polynucleotide probe )

Nr . 1 2 3 4

5 6

7 8 9 10 11

12 13 14 15 16 17 18 19 20 21 22 23 24

Strain

Isolate

ST 9 St 13 St 14 St 16 LN 60 LN 70 LN 119 LN 120 LN 463 LN 465 St 1 LN 472 St 3 St 4 St 5 St 6 pABN 1 LN 64 LN 65 LN 88 LN 357 LN 367 HB 10] pIE 745

B L B B W F B W B W

B W L

B

Crossf.assay

Hybridization radioactive digoxigenin pp OP PP

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + + +

+ + + + + + +

+ + + + + + +

+

+

+

+

L

L Contro l L L B F L Control Contro l

Identification of Aerobactin Genes in Clinical Isolates of E. coli

25

1A

00000000 ~@ O@

..

ee~

ee ~

Aerobactln-posltlv

()

Aerobactin-negatlv

B

E

• •

• •• •

.. ... •



~

• •



o

• "e

••

....

,.,

Fig. 1. Colony blot of 21 E. coli strains from different clinical isolates: The E. coli strains containing the plasmids pABN1 and pIE745 served as positive controls. E. coli HB101 was used as negative control. 1A: Schematic representation of the aerobactin production of the E. coli strains detected by the crossfeeding bioassay as well as by hybridization with radiolabelled oligo and polynucleotide probes. lB-E: Hybr idization of the strains using a digoxigenin-labelled polynucleotid e probe. B: Hybridization and detection were carr ied out as recommended by the manu facturer of the non-radioact ive DN A labelling and detection kit (Boehringer, Mannh eim). C: Additional prot einase K treatm ent of the filter dur ing prehybridization. D: Addition of blocking reagent and calf thymus DNA to the detection solution. E: Combination of proteina se K treatm ent with the modified detection procedure.

Three types of membranes were used in the non-radi oactive hybridizat ion assay nitrocellulose membran es (Serva), Imm obi lon PVDF-P- and PVDF-N membranes (M illiprobe).

26

Y. Graser and G. Schonian

At first, the colony hybridization and the detection of the hybrids were carried out as recommended by the manufacturer of the non-radioactive DNA labelling and detection kit. As shown in Fig. IB, it was impossible to distinguish between positive and negative colonies. All E. coli strains tested, i.e, the positive ones (pABNl, pIE745) as well as the negative control (HBI0l) showed hybridization signals of nearly the same intensity. A variation of the hybridization period (2, 4, 6 hours) did not improve the results (data not shown). In order to obtain reliable results, this method had to be altered as follows: a) The colonies on the filters were treated with proteinase K before hybridization. M.]. Haas and D.]. Fleming (7) obtained specific hybridization signals with biotinylated DNA probes in colony hybridization if they included a proteinase K treatment into the procedure of the preparation of colony blots. In this study, the filters were incubated 1 hour at 68°C in 20 ml of the following solution: 2 X SSC, 0.1 % SDS and 100 ug/rnl proteinase K and washed twice 15 minutes at 68°C with 20 ml2 x SSC and 0.1 % SDS. After that, the prehybridization step was carried out (Fig. 1C). b) The detection solution was altered as follows: The concentration of the blocking reagent was increased to lOA), Furthermore, calf thymus DNA was added to a final concentration of 50 ug/ml (Fig. 1D). c) The proteinase K treatment of the filters was combined with the modified detection procedure (Fig. IE). The results are shown in Fig. 1C-E. It could be clearly shown that the nonspecific attachment of the non-radioactive aerobactin probe or of the detection reagent to the lysed bacterial colonies was only avoided if, beside the proteinase K treatment of the filter, the detection protocol was also modified (Fig. IE). Among the three types of membranes tested in the non-radioactive colony hybridization assay, no significant differences were noted. Background problems were not observed. The use of gene hybridization technology in routine diagnostics for identification and characterization of bacteria is based on the development of non-isotopic hybridization assays. Because of the specific problems associated with radioisotopes, some authors have constructed DNA probes with different non-radioactive labels, i.e. biotin, N-aceroxy-N-2-acetylamino-fluorene (AAAF), digoxigenin (9). Non-radioactive colony hybridization assays yield some degree of non-specific background signals. Nylon membranes showed considerably higher background levels than did nitrocellulose membranes. Moreover, variation in background intensity and non-specific reactions between different charges of nitrocellulose membranes were observed (12). Therefore, it is necessary to include adequate controls on each batch of filter. The results obtained in the present study demonstrate that with the modification of the hybridization and detection protocols described, good agreement of the values obtained with either the radioactive or non-radioactive aerobactin gene probes was achieved. The treatment of filters with proteinase K before hybridization and a repeated blocking during the detection procedure were found to be essential to avoid non-specific probe binding. Acknowledgment. We thank Wilhelm Mann for critical advice and help in preparation of the manuscript. We are grateful to J. B. Neilands (Berkeley) for the generous gift of the plasmid pABNl and P. Buchholz (Berlin) for giving the plasmid pIE745. H. Steinruck is acknowledged for providing us with clinical samples.

Ident ificat ion of Aero bacti n Genes in Clinical Isolates of E. coli

27

References 1. Bindereif, A . and ]. B. Neila nds: Cloning of th e aerob actin-rnediated iron assimilation system of plasmid ColV. ]. Bact. 153 (1982) 1111-11 13 2. Bindereif, A. and]. B. Ne ilands: Aeroba ctin genes in clinical isolates of Escherichia coli. J. Bact. 161 (1984) 727-735 3. Boyer, H. W. and D. R oultand-D ou ssoix : Com plementation ana lysis of the restriction and mod ification of DNA in E. coli. J. Molec. BioI. 41 (1969) 459-472 4 . Buchholz, P.: Plasmidmu steran alyse von En terobacteriaceae aus seprikarnischen Infektionen der Charite. Diss, (1988) 5 . Carbo netti, N. H. and P. H. W illiams : In: The Virulence of E. coli (N . Sussma nn, ed.), pp. 419-423. Academ ic Press, Lond on-New York (1985) 6. Carbonetti, N. H., S. Bonchai, S. H. Shar ry, V. Vaisanen-Rhen, T. K. Ko rho nen, and P. H. Williams : Aerobacti n-mediated iron uptake by Escherichia coli isolates from hum an extr aintest inal infections. Infect. Immun. 51 (1985) 866-868 7. H aas, M .]. and D.]. Flemi ng : Use of biot inylated DNA pro bes in colony hybridization. Nucl . Acid Res. 14 (1986 ) 3976 8. Maniatis, T., E. F. Fritsch , and .f. Sam brook : Molecular cloning - a labor ator y manual. Cold Spring Harbor Labor ator y, Cold Spring HarborlNY (1982) 9. Ma tthews,]. A. and L.]. Kricka : Analytical strategies for the use of DN A probes . Anal. Biochem. 169 (1988) 1-25 10. Orskov, I., E. Suanborg, and F. Orsk o v: Aerobactin prod uction of seroty ped Escherichia coli from urinary tract infections. Med. Micro biol. Immun o!. 177 (1988) 9- 14 11. G raser, Y.: Her stellun g und Anwendung von Gensonden zum Na chweis des Virulenzmerkmals Aerobactin bei E. coli-Stam men aus klinischen Isolaten, Diss., Berlin (199 1) 12. Sommerfelt , H ., H. M. S. G rewal, and M. K. Bhan : Simplified and accurate nonr adio active po lynucleotide gene pro be assay for identification of enterotoxigenic Escherichia coli. ]. Clin. Micro biol. 28 (1990) 49- 54 13. Val vano, M. A. and]. H. Crosa: Aerob actin iron transport genes commonly encoded by certai n ColV plasmids occur in the chrom osome of a hum an invasive strain of Escheri chia coli K1. Infect. Immun. 46 (1984) 159- 167 14. Valvano , M. A., R . P. Silver, and H. ]. Crosa: Occurrence of chromosome- or plasmidmediated aero bactin iro n tra nspo rt systems and hemolysin production among clonal gro ups of huma n invasive strai ns of Escherichia coli Kl. Infect. Immu n. 52 (1986)

192-199

15. Valvano, M. A. and H. J. Crosa: Mo lecular cloning, expressio n and regulation in Escherichia coli K12 of a chromosome-mediated aerobactin iron tran sport system from a hum an invasive strain of E. coli Kl. J. Bact. 170 (1988) 5529-5538 16. Wi lliams, P. H .: N ovel iro n uptake system specified by ColV plasmids of invasive strains of Escherichia coli. Infect. Immun . 26 (1979) 925-932 17. W illiam s, P. H. and P.]. Warner: ColV plasmid-mediated, colicin V-independent iron uptake system of invasive stra ins of E. coli. Infect. Immu n. 29 (1980) 411-416 Dipl.-Biol. Yvo nne G raser, Institut fur Med izinische Mikrobiologie des Bereiches Medi zin (Cha rite), PF 140, D-O 1040 Berlin

Identification of aerobactin genes in clinical isolates of E. coli using a non-radioactive DNA probe.

A digoxigenin-labelled gene probe was used for the identification of aerobactin genes in 21 E. coli strains from clinical isolates by means of a colon...
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