Distribution of the antiseptic-resistance genes qacE and qacEΔ1 in Gram-negative bacteria

FEMS Microbiology Letters 159 (1998) 173^178

Distribution of the antiseptic-resistance genes qacE and qacEv1 in Gram-negative bacteria Hitoshi Kazama, Hajime Hamashima, Masanori Sasatsu *, Taketoshi Arai Department of Microbiology, Showa College of Pharmaceutical Sciences, Machida, Tokyo 194, Japan Received 24 September 1997 ; revised 1 December 1997; accepted 2 December 1997

Abstract The distribution of the antiseptic-resistance genes qacE and qacEv1 was studied in a large number of Gram-negative bacteria by a method that included the polymerase chain reaction (PCR). A total of 117 strains of Gram-negative bacteria, isolated from clinical or environmental sources, was used in this analysis. We demonstrated the presence of these genes in 48 of 78 strains of Pseudomonas, in 20 of 26 strains of Vibrio, and in four of 13 strains of other species. These results indicate that the antiseptic-resistance genes are present in a broad range of species of Gram-negative bacteria. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : qac; Antiseptic resistance; Integron; Polymerase chain reaction ; Gram-negative bacteria

1. Introduction Many kinds of antiseptic and disinfectant are used in hospitals and medical institutions and several examples of antiseptic-resistance bacteria have been reported. The resistance determinants were designated genes for resistance to quaternary ammonium compounds (qac) and small multidrug resistance genes (smr) [1]. These genes mediate resistance to DNAintercalating dyes (e.g. ethidium bromide) and to quaternary ammonium compounds (e.g. benzalkonium chloride), and it was reported that these genes encode transmembrane proteins. Thus, mechanisms of resistance encoded by the genes qacA, smr and qacE were deduced to involve e¥ux systems [1]. Al* Corresponding author. Tel.: +81 (427) 21-1552; Fax: +81 (427) 21-1593.

most all antiseptic-resistance genes, such as qacA, qacB, smr and qacF, have been isolated from Gram-positive bacteria. Only three such genes, namely, qacE, qacEv1 and emrE, have been isolated from Gram-negative bacteria to date. The genes qacE, qacEv1 and emrE were isolated from plasmid R751 of Klebsiella pneumoniae [2], the 3P conserved segment of the integron of Gram-negative bacteria [3], and the chromosomal DNA of Escherichia coli [1], respectively. It is now relatively easy to detect speci¢c genes in various bacteria by methods that involve the polymerase chain reaction (PCR). The distribution of the qacEv1 gene in bacteria has not yet been reported but several studies of the distribution of the abovementioned integron by PCR have been reported [6,7]. The integron has been found in many clinical isolates of Enterobacteria and in Pseudomonas sp.

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 5 6 3 - 6

FEMSLE 7989 5-2-98

174

H. Kazama et al. / FEMS Microbiology Letters 159 (1998) 173^178

Therefore, it seemed possible that the qacEv1 and qacE genes might also be present in many species of Gram-negative bacteria. In this study, we examined the distribution of the qacEv1 and qacE genes in many strains of Gramnegative bacteria isolated from clinical and environmental specimens using PCR.

WYOK agar (Eiken-kagaku Co. Ltd., Tokyo, Japan) for Legionella pneumophila; L-broth (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl and 1 g of glucose per liter, pH 7.0) or plates of agar-solidi¢ed L-broth that contained 1.5% (w/v) NaCl for Vibrio spp.; and Mueller-Hinton and brain heart infusion agar plates or broth (Difco Laboratory, Detroit, MI, USA) for other bacterial strains.

2. Materials and methods

2.3. Determination of drug susceptibility

2.1. Bacterial strains

Minimum inhibitory concentrations (MICs) of drugs were determined by the two-fold dilution method on Mueller-Hinton agar plates (Difco) by the standard method described by the Japan Society of Chemotherapy [4]. The drugs used were mercury dichloride (HgCl2 ), sulfamethoxazole (SA), streptomycin (SM), kanamycin (KM), gentamicin (GM), tetracycline (TC), chloramphenicol (CP) and trimethoprim (TMP). All drugs were purchased from Sigma Chemical Company, St. Louis, MO.

The strains used in this study are indicated in Table 1. Among 63 clinical isolates of Pseudomonas aeruginosa, 13, 13 and 37 strains were isolated in Japan in 1982, 1992 and 1995, respectively. The ¢ve clinical isolates of Vibrio parahaemolyticus and the seven clinical isolates of V. cholerae non-O1 were isolated in Japan in 1980. The three strains of V. alginolyticus were isolated from food in Japan in 1980. All the environmental strains were isolated in Japan in 1996. 2.2. Media The media for growth of bacteria were as follows:

2.4. Extraction of total DNA Total DNA was isolated according to a modi¢cation of a procedure reported by Sallen et al. [5]. The cells in 300 Wl of growth culture were pelleted by

Table 1 Strains examined in this study Species

Standard strains

Pseudomonas aeruginosa

JCM PAO JCM JCM

Pseudomonas putida Pseudomonas £uorescens Pseudomonas vesicularis Pseudomonas diminuta Burkholderia cepacia Xanthomonas maltophilia Alcaligenes faecalis Acinetobacter calcoaceticus Flavobacterium indologenes Legionella pneumophila Helicobacter pylori Escherichia coli Klebsiella pneumoniae Serratia marcescens Vibrio parahaemolyticus Vibrio alginolyticus Vibrio cholerae non-O1

JCM JCM JCM JCM

5516 4141 6156 5963

Environmental isolates 5

Clinical isolates 63

4 1 1 1

5506 1975 1474 6842

1 ATCC 33152 ATCC 43505, 43629 JCM 1649 JCM 1662 JCM 1239

1

10 3 1

5 7

JCM, Japanese Collection of Microorganisms (Saitama, Japan); ATCC, Americal Type Culture Collection (Rockville, MD, USA).

FEMSLE 7989 5-2-98

H. Kazama et al. / FEMS Microbiology Letters 159 (1998) 173^178

175

Fig. 1. The nucleotide sequences of the qacE and qacEv1 genes. The arrows indicate primer sequences for PCR. The asterisks show termination codons. RBS, putative ribosome-binding site; M, initiation codon.

centrifugation for 1 min at 15 000 rpm in a microcentrifuge. The bacterial pellet was washed in 300 Wl

of TE-glucose (50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA). The cells were resuspended in

Fig. 2. Lanes 1^11 show speci¢city of ampli¢cation of fragments of qacE or qacEv1 by PCR. Lane 1 was loaded with DNA size markers, PX174/HaeIII digest (Toyobo, Inc., Tokyo, Japan). Lane 2, reaction mixture after PCR with F1 and R1 primers and total DNA from E. coli JM109(pSA331) carrying qacEv1; lane 3, with F1 and R1 primers and total DNA from E. coli HB101(R751) carrying qacE; lane 4, with F1 and R2 primers and total DNA from E. coli HB101(R751) carrying qacE; lane 5, with F1 and R2 primers and total DNA from E. coli JM109(pSA331) carrying qacEv1. Lanes 6^11 are clinical isolates of P. aeruginosa carrying qacEv1, qacE or both. Lanes 6 and 7, PCR products of qacEv1 and qacE from P. aeruginosa PEK45 respectively ; lanes 8 and 9, result of the detection of qacEv1 and qacE from P. aeruginosa PEK18 respectively ; lanes 10 and 11, result of the detection of qacEv1 and qacE from P. aeruginosa PEK1 respectively. Lanes 12^17 are the qacEv1-positive strains of several Gram-negative bacteria. Lane 12, X. maltophilia JCM 1975; lane 13, S. marcescens JCM 1239; lane 14, V. parahaemolyticus ; lane 15, V. alginolyticus; lane 16, V. cholerae non-O1; lane 17, H. pylori ATCC 43504.

FEMSLE 7989 5-2-98

176

H. Kazama et al. / FEMS Microbiology Letters 159 (1998) 173^178

Table 2 Detection of qacEv1 and qacE in various Gram-negative bacteria Species

Source

P. aeruginosa

Clinical

Number of strains

1982 1992 1995

13 13 37 5 2 4 1 1 1 1 2 1 1 1 1 1 2 2 1 1 5 10 3 7 1

Environmental Standard Environmental Standard Standard Environmental Environmental Standard Standard Standard Standard Environmental Standard Standard Standard Standard Standard Clinical Environmental Environmental Clinical Environmental

P. putida P. £uorescens P. vesicularis P. diminuta B. cepacia X. maltophilia A. faecalis A. calcoaceticus F. indologenes L. pneumophila H. pylori E. coli K. pneumoniae S. marcescens V. parahaemolytica V. alginolyticus V. cholerae non-O1

Number of positive strains

300 Wl of TE bu¡er (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) and then 120 Wl of lysis bu¡er (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mg ml31 lysozyme, 100 Wg ml31 RNase A) were added with incubation at 37³C for 30 min. After incubation, 10 Wl of 10% SDS (v/w) was added with gentle mixing. An equal volume of phenol, saturated with TE bu¡er, was added with thorough mixing. This solu-

qacEv1

qacE

12 9 20 2 2 2 ^ 1 ^ ^ ^ 1 ^ ^ ^ ^ 2 ^ ^ 1 3 7 3 6 1

4 6 5 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^

tion was centrifuged for 5 min at 15 000 rpm in a microcentrifuge. The supernatant was mixed with an equal volume of chloroform. After gentle mixing, the solution was centrifuged for 3 min at 15 000 rpm. This supernatant was used for PCR. The quantity and quality of extracted nucleic acids were estimated by electrophoresis on a 0.7% agarose gel.

Table 3 MICs of antibiotics for environmental isolates of Pseudomonas sp. and some standard strains Species

P. aeruginosa JCM 5516 P. aeruginosa 516 P. aeruginosa 563 P. putida 519 P. putida 560 P. putida 562 X. maltophilia JCM 1975 S. marcescens JCM 1239

MIC (Wg ml31 )

PCR

HgCl2

SA

SM

12.5 6.25 90.1 12.5 90.1 90.1 25 25

v1600 v1600 v1600 v1600 v1600 v1600 50 1600

100 400 50 1.56 12.5 0.78 50 1.56 25 1.56 100 v1600 3.13 1.56 3.13 6.25

KM

GM

TC

CP

TMP

qacEv1

qacE

12.5 1.56 0.78 1.56 1.56 12.5 0.2 1.56

100 6.25 6.25 12.5 6.25 100 90.1 100

800 800 50 800 100 1600 0.2 12.5

800 400 400 400 400 400 3.13 3.13

+ + + + + + + +

3 3 3 3 3 3 3 3

+, positive PCR; 3, negative PCR.

FEMSLE 7989 5-2-98

H. Kazama et al. / FEMS Microbiology Letters 159 (1998) 173^178

2.5. Primers for PCR and control plasmids The primer was synthesized by Griner Japan (Akasaka, Minatoku, Tokyo, Japan). The nucleotide sequences of primers were as follows: forward primer for ampli¢cation of qacE and qacEv1 (F1), 5PGGGAATTC-(2225)-GCCCTACACAAATTGGGAGA-(2244)-3P; reverse primer for qacEv1 (R1), 5PGCTGCAG-(2586)-CTGCGGTACCACTGCCACAA-(2605)-3P; and reverse primer for qacE (R2), 5PTACTCGAG-(5093)-TTAGTGGGCACTTGCTTTGG-(5112)-3P. The numbers in the nucleotide sequences of qacE and qacEv1 refer to the sequences in the EMBL/GenBank/DDBJ Nucleotide Sequence Data Libraries with accession numbers X72853 and D43625, respectively. The standard control template DNAs were total DNA isolated from E. coli HB101(R751) [2] for qacE and total DNA isolated from E. coli JM109 (pSA331) [6] for qacEv1. 2.6. PCR For PCR, we used the Takara Taq kit or the Takara Ex Taq kit (Takara Shuzo Co. Ltd., Shiga, Japan). PCR was performed with 30 cycles of steps as follows: denaturation at 94³C for 1 min, annealing at 50³C for 1.5 min and extension at 72³C for 1 min. The products of PCR were detected by electrophoresis on a 1.5% agarose gel.

3. Results The nucleotide sequences of qacE and qacEv1 are very similar (the extent of homology is about 80%). Therefore, the primers were designed to distinguish between these genes, as shown in Section 2 and Fig. 1. Ampli¢ed products of qacE and qacEv1 DNA are shown in Fig. 2. The results of our experiments are summarized in Fig. 2 and Table 2. Among the 63 clinical isolates of P. aeruginosa, qacEv1 and qacE were detected in 41 and 15 strains, respectively. The qacE gene was detected only in clinical isolates of P. aeruginosa. Fourteen of 15 strains with the qacE gene also possessed the qacEv1 gene. Four strains of Pseudomonas isolated from the environment were positive for the qacEv1 gene but the strains of P. vesicularis, and

177

P. diminuta were negative for the qacEv1 gene (Table 2). The strains of Serratia marcescens and Xanthomonas maltophilia tested were also positive for the qacEv1 gene. Three clinical isolates and seven environmental isolates of V. parahaemolyticus, three environmental isolates of V. alginolyticus and six clinical isolates and one environmental isolate of V. cholerae non-O1 were positive for the qacEv1 gene. Among the other bacteria examined, two strains of Helicobacter pylori tested were also positive for qacEv1 (Table 2). The susceptibility to antimicrobial agents was determined for the strains isolated from the environment and for standard strains that were positive for qacEv1. All strains tested were resistant to sulfonamide (Table 3).

4. Discussion A large number of Gram-negative bacteria, including many strains of Pseudomonas sp., were tested for the presence of the qacE and qacEv1 genes. The percentage of strains positive for qacEv1 and qacE among the clinical isolates of P. aeruginosa was 68% and 24%, respectively. The qacEv1 gene is located in the 3P conserved segment of the integron. Leèvesque et al. reported that 24 of 35 clinical isolates of Pseudomonas spp. and Enterobacteria harbored an integron [5,7]. The results of our experiments correspond to those in the report by Leèvesque et al. Almost all of the qacE-positive strains were also positive for qacEv1. This result indicates that these strain had two kinds of integron, one with qacE and one with qacEv1 in the 3P conserved segment. We examined the susceptibility to sulfonamide of some strains that were positive for qacEv1. All qacEv1-positive strains were resistant to the drug. The qacEv1 gene probably represents a disrupted form of qacE that evolved by insertion of a DNA segment containing a sulfonamide-resistance gene near the 3P end of the qacE gene [2]. Only one strain of P. aeruginosa, isolated from a clinical specimen in 1995, harbored the qacE gene. Therefore, the integron with no 3P conserved segment was present in few clinical isolates of P. aeruginosa. Our results indicate that the sulI gene is strongly linked to the integron [7]. In the strains of P. aeruginosa isolated from en-

FEMSLE 7989 5-2-98

178

H. Kazama et al. / FEMS Microbiology Letters 159 (1998) 173^178

vironmental specimens, two strains had the qacEv1 gene. These two positive strains were resistant to several antimicrobial agents, such as SA, CP, TMP or SM (Table 3). The integron has mainly been detected in clinical isolates, but we found evidence of the integron in environmental bacteria. Other positive strains were of P. putida and P. £uorescence. The environmental isolates of P. putida with the qacEv1 gene were also resistant to SA, CP or TMP (Table 3). The qacEv1 gene might be widely distributed among Pseudomonas spp. The qacEv1 gene was detected in strains of Helicobacter and Vibrio. The qacEv1 genes from Helicobacter and Vibrio have been cloned, and are now being studied in greater detail.

References

[2]

[3]

[4] [5]

[6]

[7]

[1] Paulsen, I.T., Brown, M.B. and Skurray, R.A. (1996) Proton-

dependent multidrug e¥ux systems. Microbiol. Rev. 60, 575^ 608. Paulsen, I.T., Littlejohn, T.G., Raîdstroëm, P., Sundstroëm, L., Skoëld, O., Swedberg, G. and Skurray, R.A. (1993) The 3P conserved segment of integrons contains a gene associated with multidrug resistance to antiseptics and disinfectants. Antimicrob. Agents. Chemother. 37, 761^768. Stokes, H.W. and Hall, R.M. (1989) A novel family of potentially mobile DNA elements encoding site-speci¢c gene-integration functions : integrons. Mol. Microbiol. 3, 1669^1683. Japan Society of Chemotherapy (1981) Chemotherapy. 29, 76^79. Sallen, B., Rajoharison, A., Desvarenne, S. and Mabilat C. (1995) Molecular epidemiology of integron-associated antibiotic resistance genes in clinical isolates of Enterobacteriaceae. Microbial Drug Resist. 1, 195^202. Kazama, H., Kizu, K., Iwasaki, M., Mamashima, H., Sasatsu, M. and Arai, T. (1995) Isolation and structure of a new integron that includes a streptomycin resistance gene from the R plasmid of Pseudomonas aeruginosa. FEMS Microbiol. Lett. 134, 137^141. Leèvesque, C., Piche, L., Larose, C. and Roy, P.H. (1995) PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39, 185^191.

FEMSLE 7989 5-2-98