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Transferable plasmid-mediated antibiotic resistance in Acinetobacter
PLASMID
10,
138-147 (1983)
Transferable
Plasmid-Mediated
Antibiotic
Resistance
in Acinetobacter
F. W. GOLDSTEIN,*A. LABIGNE-ROLJSSEL,t*’ G. GERBAUD,? C. CARLIER,~ E. COLLATZ,~” AND P. CouRvALrNt*3 *Laboratoire de Mcrobiologie Mt!dicale, H6pital St. Joseph, 75014 Paris, and TLaboratoire de Biochimie, L..4. CNRS 271. UnitP de Bactkiologie Midicale. Institut Pasteur, F-75724 Paris, France
Received March 16. 1983 Acinetobacter calcoaceticus strain BM2500 was resistant to ampicillin. aminoglycoside-aminocyclitols, chloramphenicol, sulfonamides, and high levels of trimethoprim. Resistanceto ampicillin was due to the presenceof a /3-lactamase(TEM-I) and the aminoglycoside-aminocyclitol resistancewas mediated by phosphotransferase(APH(3’)(5”)1)and adenylyltransferase(AAD(3X9)) activities. The resistance genes were carried by a 167 kilobase plasmid, pIPlO I, belonging to incompatibility group 6-C; the plasmid was self-transferable, at extremely low frequency, to Escherichia co/i by conjugation. Plasmid pIPlO 1 DNA wasanalyzed by agarosegel electrophoresis following restriction endonucleasedigestion, by nucleic acid hybridization, and by CsCl analytical density gradient ultracentrifugation. The results support the hypothesis that plasmid PIP 1031 may have been acquired recently by strain BM2500.
Acinetobacter is a ubiquitous Gram-negative, aerobic, nonfermentative coccobacillus increasingly responsible for nosocomial infections and resistant to antibiotics, in particular penicillins, aminoglycoside-aminocyclitols, chloramphenicol, and tetracyclines (Bergogne-Birezin et al., 197 1; Murray and Moellering, 1979). Acinetobacter is naturally resistant to cephalosporins due to the production of a cephalosporinase (Morohoshi and Saito, 1977) and to low levels of trimethoprim (with minimal inhibitory concentrations (MICs) of 16 to 32 &ml). In certain strains, resistance to penicillins (Devaud et al., 1982), aminoglycosideaminocyclitols (Gomez-Lus et al., 1980; Le Goffic and Martel, 1974; Dowding, 1979; Murray and Moellering, 1979, 1980; Bergogne-Birezin et al., 1980; Shimizu et al., 1981; Devaud et al., 1982) and chloramphenicol (Devaud et al., 1982) is due to en’ Presentaddress:Department of Medical Microbiology. Stanford University School of Medicine. Stanford, Calif. 94305. Z Present address:Laboratoire de Microbiologic, UER 82 Les Cordeliers, Universite de Paris VI, 75270 Paris Cedex 06. 3To whom reprint requests should be addressed. 0147-619X/83 $3.00 Copyright @ 1983 by Academic Press. Inc. All nghls of reproduction in any form re%rVed.
zymes which modify these antibiotics. Suggestive evidence of plasmid-mediated resistance toward penicillins, aminoglycosides, tetracyclines, sulfonamides, and mercury has recently been reported (Murray and Moellering, 1980) and transfer of sulfonamide resistance has been obtained between strains of A. calcoaceticus (Hinchliffe and Vivian, 1980). Resistance (R) plasmid transfer from a clinical isolate of A. calcoaceticusto Escherichia coli has been reported (Gomez-Lus et al., 1980). In this study we describe a strain of A. calcoaceticus subsp. anitratus resistant to penicillins, aminoglycoside-aminocyclitols, chloramphenicol, sulfonamides, and high levels ( 1 mg/ml, or higher) of trimethoprim. The resistancegenes(R determinants) are carried by a plasmid, belonging to incompatibility (Inc) group 6-C. The mechanisms of resistance toward P-lactams and aminoglycoside-amino4 Abbreviations used: ColE I, colicinogenic factor E I ; kb, kilohasc; R plasmid. antibiotic resistanceplasmid; R determinant, antibiotic resistancemediating gene: cRNA. complementary RNA: SIOO,supematant (100,OOOg)of a bacterial extract after sonication; genotypes include aad(33(9). aminoglycoside-aminocyclitol adenylyltransferase3”. 9; aph(3’)(5”), aminoglycoside phosphotransferase Y.5”.
138
139
RESISTANCE PLASMID IN Acinefobacfer
cyclitols are described and the plasmid R determinants are compared with those of known transposons. An exogenous origin for the R plasmid is proposed. MATERIALS
AND METHODS
Bacterial strains. The sourcesand properties of the bacterial strains are listed in Table 1. Media. Tryptic soy broth and agar (Difco) were used. Disc sensitivity tests were done on Mueller-Hinton agar. All incubations were at 37°C. Genetic techniques.Conjugation (Chabbert, 1957) transformation (Davis et al., 1980), determination of incompatibility (Chabbert et al., 1972) and curing of antibiotic resistance traits (Bouanchaud et al., 1969) were performed as described. Assa)?.for aminoglycoside-aminocyclitolmodifying enzymes. The enzymes were assayedas described (Haas and Dowding, 1975).
Preparation ofDNA. High-molecular weight (Labigne-Roussel et al., 1981) and low-molecular weight (Davis et al., 1980) plasmid DNA was purified as described. DNA from bacteriophage Xc1857and derivatives (Courvalin and Fiandt, 1980) and A. calcoaceticus high-molecular weight total DNA (Roussel and Chabbert, 1978) were prepared as described. Agarose gel electrophoresis. DNA was digested with restriction endonucleases in 100 mM Tris-HCl (pH 7.8) 100 tYtM MgC12 for EcoRI and in 6 tnM Tris-HCl (pH 7.0). 6 mM MgC12, 50 mM for HindIII. Agarose gel electrophoresis was as described by Shinnick et al. (1975). Hybridization. 32P-labeledDNA (Maniatis et al., 1975)and complementary RNA (cRNA: Courvalin and Fiandt, 1980) were prepared as described. Restriction endonuclease-generated DNA fragments fractionated by agarose
TABLE I PROPERTIES OF THE BACTERIAL
Number
STRAINS USED
Relevant characteristics and plasmid content’
Strain
Source or reference
BM15
E. coli K12
azi rpoB pro met
Spontaneous mutant of 553 (Clowes and Hayes, 1968)
LA101
E. coli K12
str.4 hsdR- sulI sulll
Derived from C600 (Pourcel et al.. 1979)
BM694
E. coli C
nal.4
Spontaneous mutant of Cla (Labigne-Roussel et al.. 1981)
BM2500
A. calcoacericus
pIPlO Tra+ Inc 6-C Ap Cm Km Sm Su Tpb pIP1032, pIP1033, pIPlO34, pIP1035,pIP1036
Wild strain
BM2500- 1
A. calcoaceticus
pIP1032, pIP1033, pIP1034. pIP1035. pIPlO
Curing of BM2500
BM250 I
E. co/i K12
azi rpoB pro met
Conjugation BM2500 X BM I5
PIP 103I Tra+ Inc 6-C Ap Cm Km Sm Su Tp BM2502
E. co/i K12
str.4 hsdR- sull &II
Conjugation BM2501 X LA101
pIPlO Tra+ Inc 6-C Ap Cm Km Sm Su Tp BM2503
E. coli C
nal.4
pIPlO Tra+ Inc 6-C Ap Cm Km Sm Su Tp ’ Genetic symbols are according to Bachmann et al. (1976) and Novick ’ Designation of plasmid and its phenotypic characters are aligned.
Transformation PIP 103I X BM694
et a/. (I 976).
140
GOLDSTEIN ET AL.
gel electrophoresiswere transferred to cellulose nitrate sheets (Sartorius) as described by Southern (1975). Isoelectric.focusing
in polyacrylamide
gels.
Isoelectric focusing of S100 preparations was as described (Matthew et al.. 1975). DNA base composition. DNA base composition was determined after buoyant density measurements as described (Courvalin et al.. 1974). Enzymes. Restriction endonuclease EcoRI (Siimegi et al., 1977)was purified as described.
Endonuclease Hind111was kindly provided by J. Gardner and R. Jorgensen. Endonuclease BarnHI, from Boehringer, was usedaccording to the manufacturer’s recommendation. Enzyme sources were proteinase K (Merck), lysozyme (Sigma), ribonuclease A (bovine pancreas) (Calbiochem), and E. coli K12 RNA polymerase holoenzyme (Miles). Chemicals. [ l-‘4C]acetyl-coenzyme A ([ “C]CoASAc), adenosine 5’-[a-32P]triphosphate, triethylammonium salt ([a-32P]ATP), adenosine 5’-[y-3’P]triphosphate, triethylammonium salt ([T-~‘P]ATP), and [U“C]adenosine 5’-triphosphate, ammonium salt ( [U-14C]ATP) were obtained from the Radiochemical Centre, Amersham, England. The antibiotics were provided by the following laboratories: gentamicins (Gen) A and B, Schering; Kanamycins (Kan) A, B, C, and amikacin (Ami), Bristol; neomycin B (NeoB), spectinomycin (Spc), Upjohn; paromomycin (Par) and butirosin (But), Parke-Davis; tobramycin (Tob). Lilly: lividomycin (Liv) A, Kowa; ribostamycine (Rib), Meiji: streptomycin (Str), Pfizer; chloramphenicol, Roussel; rifampin. Lepetit; nalidixic acid, Winthrop: trimethoprim, Roche. Sarkosyl (sodium lauryl sarcosinate) was provided by Colgate-Palmolive.
methoprim (Tp) (Table 1). In curing experiments these characters were lost en bloc (approximately 0.3% of 300 colonies tested) and one cured strain, BM2500- 1, was studied further. The genes conferring resistance to ApCmKmSmSuTp were transferred with a very low frequency, about 5 X lo- lo, from BM2500 to E. cofi BM 15 by conjugation (Table 2). These R determinants were further transferred from one transconjugant (strain BM2501, Table 1) to E. cofi LA 101 by conjugation. Selection for transfer of any of these resistance characters (Table 2) revealed cotransfer of all R determinants (in 10 clones studied). One strain, BM2502 (Table l), was selected for further studies. In this case, the frequencies of transfer were 3 to 8 X 10m6 (Table 2). Incompatibility testing of the transconjugant BM2501 revealed that all the acquired characters were borne by a single plasmid belonging to the incompatibility (Inc) group 6C (Chabbert et al., 1972; Datta, 1975). Plasmid Content of BM2500, Its Derivative BM2500-1. and Transconjugant BM2501
The plasmid DNA from strains BM2500, BM2500- 1, and BM250 1 was purified by ultracentrifugation and analyzed by agarosegel electrophoresis, before (Fig. 1, Table 1) and after (data not shown) digestion with EcoRI endonuclease. Comparative analysis of the phenotypes with the plasmid content and the EcoRI-genTABLE 2 FREQUENCYOF PLASMIDTRANSFER
Donor
Recipient
Selected resistance from donor
RESULTS
BM2500
BM15
TP
Plasmid-Mediated Characters Expressed bJ A. calcoaceticus BM2500
BM250 I
LA101
SP Cm Km TP
Frequency” 5 x IO-‘” 8x 3.2 x 5x 7.3 x
Io-c 1o-6 1o-6 1o-6
Acinetobacter calcoaceticus BM2500 encodes resistance to ampicillin (Ap), chlora Frequenciesare expressedrelative to 2 X 10m9bacterial amphenicol (Cm), kanamycin (Km), strep- donors after mating on cellophane membranes (Chabbert, tomycin (Sm), sulfonamide (Su), and tri- 1957) for 18 h.
RESISTANCE BM 2500-l
BM 2500
PLASMID
BM 2501
-11 6.3 7.6
e 6.3
5.5
e 5.4
4.6 I
4.4
FIG. 1. Analysis of plasmid DNA. Plasmid DNA was fractionated by electrophoresis in a 0.6% agarose gel ( 18 X 13 X 0.4 cm) for 18 h at 3 V/cm. Right, plasmids pBR322, 4.4 kb, pBR325, 5.4 kb; ColEI, 6.3 kb; and pSF2124, I 1 kb, were used as molecular size standards. Left, the size of the plasmids is expressed in kilobases. Plasmids pIPlO (upper) and pIPlO (lower) are indicated by white arrows.
141
IN Acinetobacter
erated fragment patterns of plasmid DNA in the individual strains led us to conclude that the wild-type strain BM2500 harbors six plasmids (Table 1). Plasmid pIPlO encoded all the transferable resistances;it had a molecular size of approximately 167 kilobases (kb) and eight EcoRI-generated DNA fragments which were numbered in order of decreasing size. The other plasmids were cryptic. Plasmids pIP1032, pIP1033, pIP1034, pIP1035, and PIP 1036 had molecular sizesof about 70, 8.3, 7.6, 5.5, and 4.6 kb, respectively. Strain BM2500-1 was susceptible to ApCmKmSmSuTp and had lost pIPlO 1. E. cofi strain BM2501 was resistant to ApCmKmSmSuTp after acquisition of PIP 1031. Plasmid pIPlO was detected in BM2502, after conjugation, and in BM2503, after transformation (Table
1).
Mechanisms of Plasmid-Mediated Resistance to Antibiotics in BM2500 Strain BM2500, its derivative BM2500- 1, and the transconjugant BM2501 (Table 1)
A.calcoaceticus E. ___coli __- - . 812501
112500
_---. 1 FIG. 2. Substrate profiles of enzymes extracted from A. calcoaceficusstrain BM2500 (solid lines) and E. (plain area) or adenylylation (hatched area) are expressed relative to neomycin B or streptomycin as 10096, respectively. No acetylation of neomycin B, streptomycin, or spectinomycin, no phosphorylation of streptomycin or spectinomycin, and no adenylylation of neomycin B were detected in these strains. No aminoglycoside-modifying activity was detected in the susceptible strain BM2500- 1.
coli strain BM250 I (dashed lines). Phosphorylation
142
GOLDSTEIN ET AL.
were examined for aminoglycoside-aminocyclitol-modifying activities. Strains harboring plasmid PIP 103I were found to contain aminoglycoside phosphotransferase (APH) and aminoglycoside-aminocyclitol adenylyltransferase(AAD) but no acetyltransferase (AAC) activities. The substrateprofiles of the enzymes extracted from strains BM2500 and BM250 1 are shown in Fig. 2. The fact that kanamycin B was phosphorylated and tobramycin not, indicates that the 3’-hydroxyl group is the site of phosphorylation (APH(3’)). That lividomycin A is a substrate for phosphorylation but butirosin not, indicates that the enzyme is of type I (APH(3’)(5”)1) (Davies and Smith, 1978). The adenylylating enzyme is equally effective against streptomycin and spectinomycin, thus probably an (AAD(3”)(9)) (Davies and Smith, 1978). Strain BM2500 is resistant to penicillins and cephalosporins. Escherichia coli BM250 I is resistant to penicillins but not to cephalospotins and A. calcoaceticusBM2500- I is resistant solely to cephalosporins. Strain BM2500 encodestwo P-lactamaseswith isoelectric points of about 9.5 and 5.4 (Fig. 3). The former enzyme, also present in BM2500- 1, should correspond to the chromosome-mediated cephalosporinase (Morohoshi and Saito, 1977). The latter enzyme, also detected in BM2501 is a TEM- 1 penicillinase encoded by PIP 1031. We did not find any inactivation of chloramphenicol by strains BM2500 and BM250 I using a microbiological detection technique (Goldstein et al., 1977). Analysis of DNA bv Hybridization Total DNA from BM2500 and pIPlO DNA were hybridized to plasmid or bacteriophage probes (Fig. 4). The properties of the template DNAs used to localize the R determinants and to search for the presence of insertion sequences(IS) are summarized in Table 3. In each experiment, the reaction with the pIPlO 1 probe was used as a control. No differences were observed between the hybridization patterns of PIP 103I (Fig. 4B) and BM2500 total DNA (Fig. 4C). The kanamycin R determinant was localized with X::Tn601 and X probes on EcoRl
.w
-C
,:!:i ‘,,I
_ 5.6 - 5.4
-----_ FIG.3. Analytical isoelectricfocusing.&lactamases from the strains indicated on the top were focusedand revealed by nitrocefm (Glaxo). 1, E. coii K12/pSF2124 encoding a TEM-I &lactamase (PI = 5.4): 2, E. co/i K12/RP4 encoding a TEM-2 j3-lactamase(PI = 5.6): C. cephalosporinase.
fragment 4 of pIP 103I. Transposableelements Tn6 (Berg et al., 1975) and Tn601 (Davies et al., 1977) later designated Tn903 (Table 3) confer resistance to kanamycin by synthesis of APH(3’)(5”)-I enzymes with substrate profiles similar to that encoded by pIPlO (Fig. 2). The phosphotransferase genes in the two transposons are related (Courvalin et al., 1979). Both Tn6 and Tn602 are composite classI elements (Kleckner, 1981) in which the region encoding kanamycin resistance is flanked by insertion sequences(IS), IS903 in the caseof Tn601 (Grindley and Joyce, 1980) and IS15 (Labigne-Roussel and Courvalin, 1983) in the case of Tn6. Both X::Tn6 and X: :Tn60Z hybridized to EcoRI fragment 4 of pIPlO 1 and X: :Tn6 hybridized, in addition, to fragment 3. Hybridization experiments (Labigne-Roussel and Courvalin, 1983) re-
143
RESISTANCE PLASMID IN Acinetobacler
PROBES
,
plP1031
(*I
(6)
BM2500
Total
DNA
‘j
DRIVERS (EcoRI)
@I
FIG. 4. Analysis of DNA by hybridization. Plasmid pIPlO I DNA or BM2500 total DNA (drivers), were digested with EcuRI. The resulting fragments were fractionated by gel electrophoresis (0.7% agarose, 18 X 13 X 0.4 cm) for 12 h at 3 V/cm, transferred to nitrocellulose sheets, and hybridized to in vitro 32Plabeled DNA (or cRNA for ColE 1::Tn7*) (probes). The size of the eight PIP 1031 EcoRI-generated DNA fragments (A) was determined using Xc1857 DNA, 48 kb, digested with EcoRI, SmnI, and EcoRI + Hind111 as molecular size standard. Bands 5 to 8 in (C), although faint on the photograph were clearly visible on the autoradiogram.
vealed the presence of a natural deletion derivative of ISIS, ISI5A, in pIPlO EcoRI fragments 3 and 4. Plasmid PIP 1031 encodes a TEM- 1 fl-lactamase (Fig. 3). The pSF2124 (Ap) probe hybridized to fragment 3 and, although with a weaker intensity, also to fragment 4 of PIP 1031. The ubiquitous Tn3 element does not contain an EcoRI site. In pIPlO 1, we cannot discriminate between the presence of two ampicillin R determinants or the occurrence of an EcoRI site in its P-lactamasegene. The EcoRI (SmSu) insert in pCH 13 allowed us to assignthe streptomycin and sulfonamide determinants to EcoRI fragments 3 and 6 of pIPlO 1. As in the case of ampicillin resistance, we do not know if there is an EcoRI site in the Sm or Su R determinants of pIPlO 1 or if there is a duplication of either or both genes. A TEM-1 gene possessingan
EcoRI site and a plasmid carrying two related AAD(3”)(9) genes have been described (Iabigne-Roussel et al., 1982). The transposable element Tn7 confers resistance to trimethoprim by synthesis of a dihydrofolate reductase type I (Pattishall et al., 1977) and to streptomycin by synthesis of an AAD(3”)(9). We detected homology between ColE 1: :Tn7 DNA labeled by nick-translation and pIPlO 1 EcoRI fragments 3 and 6 but not between a ColE 1: :Tn 7 cRNA probe and pIPlO 1. While the dfr1 gene is transcribed under the conditions used, the aad gene is not (Iabigne-Roussel et al., 1982). These results agreewith the existence, on pIPlO 1, of either two homologous Sm R determinants or the occurrence of an EcoRI site in the region encoding the adenylylating enzyme; in addition, they indicate that resistance to trimethoprim in BM2500 is not due to a dihydrofolate re-
144
GOLDSTEIN ET AL. TABLE 3 PLASMIDS
AND BACTERIOPHAGES
Plasmid or bacteriophage ColEl pSF2I24 pCH13’ ColE I : :Tn7 xc X::Tn6d X::Tn601’ X::ISI’ PlCm
USED AS PROBES FOR HYBRIDIZATION
Relevant genotype”
Source or reference
-
Bazaral and Helinski (1972) So et al. ( 1975) J. Davies N. Datta Fiandt ef al. (I 977) Berg er al. (I 975) Davies et al. (I 977) P. Starlinger Kondo et al. ( 1964)
bla (TEM- 1)
ad (3”)(9), sul aad (Y)(9). dfr1 aph (3’)(Y)-1 uph (3’)(5”)-I
cat
’ Genetic symbols are according to Novick et al. ( 1976) and Davies and Smith ( 1978). bpCH13, ColElQ {r&(3”)(9), sul). ‘A, XcI857. d X::Tn6, hb5 1565I9 ~I857 Sam7::Tn6. kindly provided by R. Jorgensen. ‘X::Tn60l, Xb515b519 c1857 .Sam7::Tn601.
’ h::ISI, XI, Xdgur ~1857S~68-0p306::ISI, kindly provided by P. Starlinger.
ductase closely related in sequence to that of Tn7. No hybridization was detected between pIPlO and Xc1857,ColEl, X::ISZ, or PlCm. The latter is consistent with our failure to detect inactivation of chloramphenicol by strains BM2500 and BM2501.
icol, kanamycin, streptomycin, sulfonamides, and trimethoprim in A. calcoaceticus subsp. anitratus strain BM2500 are located on a 167 A I
Base Composition of DNA Chromosomal and plasmid DNA from BM2500 were analyzed by CsCl analytical density gradient ultracentrifugation (Fig. 5). The buoyant density of the chromosomal DNA was 1.6907, corresponding to a G + C content of 37.5%. The buoyant density of pIPlO 1 was 1.700 which corresponds to 46.5%G + C. When plasmid DNA from strain BM2500- 1 was analyzed, a peak and a shoulder were seen. The peak corresponding to a density of 1.693 (39.5% G + C) should represent PIP 1032; the shoulder, corresponding to a density of 1.69 (G + C 36.5%) should represent the pool of the small molecular size plasmids pIP1033, pIPlO34, pIP1035, and pIP1036. DISCUSSION We have demonstrated that the genesconferring resistanceto ampicillin, chloramphen-
:’
---#I---
PIP
1031
1 I -
-J
BY 2500.1 ptm8mld DNA
ii ---
-.J
BM 2500 total DNA 1.703
1.700
1.6907
FIG.5. Analysis of DNA by isopycnic ultracentriiirgation in CsCI. Bacteriophage Xc1857DNA (p = 1.703 g/cm-‘) was included as an internal standard.
RESISTANCE PLASMID IN Acinetobacter
kb plasmid designated pIPlO 1. The loss or acquisition of this resistancephenotype is correlated with the absenceor presenceof plasmid pIPlO DNA (Fig. 1). In strain BM2500 resistance to streptomycin and spectinomycin is mediated by a 3” 9-adenylyltransferase (AAD(3”)(9)) and resistanceto kanamycin and structurally related compounds is due to the synthesis of a 3’5” phosphotransferase of type I (APH( 3’)(S’)-I) (Fig. 2). Enzymes with identical site specificity have been described in Acinetobacter (Bergogne-B&-&n et al., 1980; Gomez-Luz, 1980; Murray and Moellering, 1980; Shimizu et al., 1981; Devaud et al.. 1982). Resistanceto ampicillin, carboxy- and ureidopenicillins is due to the presenceof a TEM- 1 /I-lactamase (Figs. 3 and 4). Resistance of strain BM2500 to chloramphenicol does not appear to take place by inactivation of the antibiotic; resistance to sulfonamide is also by an unknown mechanism. Resistance to trimethoprim is novel in Acinetobacter but the biochemical mechanism for this resistance remains unknown. As inferred from hybridization experiments (Fig. 4) it is not due to a dihydrofolate reductase of type I. Incidentally. a dihydrofolate reductase of type II has been found associatedwith an Inc 6-C plasmid (seebelow) isolated in the same hospital as pIPlO 1 (Pattishall et al., 1977). The G + C content of BM2500 chromosomal DNA is 37.5% (Fig. 5) a ratio expected for a member of this species(Normore, 1976). This value, which represents an average for the whole genome, is rather close to those (39.5 and 36.5%) estimated for the cryptic plasmids of this strain. However, the base composition of PIP 1031 is 46.5% G + C which presumably indicates a foreign origin of this replicon. Plasmid pIPlO 1 is self-transferable by conjugation, at extremely low frequency (Table 2) to E. co/i. Curiously, this transfer could only be obtained with the initial isolate of A. calcoaceticus BM2500. As judged from the EcoRI fragment pattern of BM2500 plasmid DNA, and from hybridization between the total DNA of initial or subsequent cultures of this strain and a PIP 1031 probe (Fig. 4) the
145
loss of this property was not due to major rearrangements in the plasmid genome nor to integration of the R determinants into the host chromosome. This phenomenon leads us to speculate that plasmid PIP 1031 was acquired only extremely recently by strain BM2500. This is in contrast with the situation in E. coli where pIPlO 1 is stable and can be easily transferred by conjugation (Table 2) or transformation (Table 1). Plasmid pIPlO belongs to the incompatibility group 6-C (Chabbert et al., 1972). Like Inc P, and to a lesser extent Inc 7-M plasmids, plasmids of this group are ubiquitous. They are frequently encountered in France (J. Witchitz, personal communication) especially in Enterobacteriaceae (Acar et al., 1977). They also possessa wide host range and have been found in many diverse pathogenic bacterial species including rleromonas hydrophila (Mizon et al., 1978), Pseudomonas aeruginosa (Chabbert et al., 1972) and Vibrio choferue (Rahal et al., 1973). This broad host range may be related to the fact that the DNA of the Inc 6-C plasmids is relatively inert to restriction endonuclease digestion. For example, EcoRI (Fig. 4) HindIII, BumHI, and Pst 1 (J. Witchitz, personal communication and our unpublished data) generateonly a few large DNA fragments. Becausethese plasmids are extremely common and can easily cross generic barriers, the Inc 6-C plasmids are likely vectors for the introduction of new genetic information to genera like Acinetobacter, which is known to produce a large variety of restriction endonucleases(Roberts, 1982).The same seemsto be true for the Inc P plasmids which have been found to transfer resistance genes from clinical isolates of Acinetobacter to E. cofi (Gomez-Lus et al., 1980) or from E. coli to .kinetobacter (Towner and Vivian, 1976). The -4. calcoaceticus strain BM2500 was isolated from a clinical specimen at the Saint Joseph Hospital in Paris. In this ecosystem, the ApCmKmSmSuTp determinants are commonplace (F. Goldstein, unpublished observation) and are most frequently (i.e., in about 40% of the cases)borne by Inc 6-C plasmids (Acar et al., 1977;Goldstein et al.. 1982).
146
GOLDSTEIN
ET AL.
We then consider the presence of plasmid COURVALIN,P. M., CARLIER,C., CROISSANT,0.. AND BLANGY,D. (1974). Identification of two plasmids dePIP 1031 in a wild strain of Acinetobacter as termining resistanceto tetracycline and to erythromycin an indication that C type plasmids have an in group D Streptococcus. Mol. Gen. Genet. 132, 18 I even broader host range than was suspected. 182. DATTA, N. (1975). Epidemiology and classification of plasmids.In “Microbiology-1974” (D. Schlessinger.ed.). ACKNOWLEDGMENTS pp. 9-15. Amer. Sot. Microbial. Washington, D. C. We thank A. Fritsch for buoyant density determinations, DAVIES, J., BERG, D., JORGENSEN,R., FIANDT, M.. 0. Rouelland for secretarialassistance,and Y. A. Chabbert HUANG, T. S. R.. COURVALIN,P., AND SEHLOR‘,J. for material support and otherwise. (1977). Transposable neomycin phosphotransferases.In “R-factors: Their Properties and Possible Control” (J. REFERENCES Drews and G. Hogenauer, eds.) pp. 10I - I IO. SpringerACAR, J. F., GOLDSTEIN,F. W., GERBAUD,G. R., AND Verlag, Wien, New York. CHABBERT,Y. A. (1977). Plasmides de resistance au DAVIES,J.. AND SMITH, D. I. (1978). Plasmid-mediated trimethoprime: transferabilite et groupes d’incomparesistanceto antimicrobial agents.Annu. Rev. Microbial. tibilite. Ann. Microbial. (Inst. Pasteur) 128A, 4 l-47. 32,469-5 18. BACHMANN, B. J., BROOKSLow, K.. AND TAYLOR, DAVIS, R. W., BOTSTEIN,D., AND ROTH, J. R. (1980). A. L. (1976). Recalibrated linkage map of Escherichia “Advanced Bacterial Genetics,” p. 140. Cold Spring coli K 12. Bacterial. Rev. 40, I 16- 167. Harbor, N. Y. BAZARAL,M., AND HELINSKI, D. R. (1972). Character- DEVAUD, M.. KAYSER. F. H., AND BACHI, B. (1982). ization of multiple circular DNA forms of colicinogenic Transposon-mediated multiple antibiotic resistance in factor E I from Proteus mirabilis Biochemistry 7, 35 I3Acinetobacter strains. Antimicrob. Agents Chemother. 3519. 22, 323-329. BERG, D. E.. DAVIES. J.. ALLET, B., AND ROCHAIX, DOWDING,J. E. (1979). Novel aminoglycoside modifying J. D. (1975). Transposition of R factor genes to bacenzyme from a clinical isolate of Acinetobacter. J. Gen. teriophage X. Proc. Nat. .Acad. Sci. USA 72,3628-3632. Microbial. 110, 239-24 I. BERGOGNE-BEREZIN, E., JOLY.M. L., MOREAU,N., AND FIANDT, M., HONIGMAN, A., ROSENVOLD,E. C., AND LE Gomc, F. (1980). Aminoglycoside-modifying enSZYBALSKI.W. ( 1977). Precise measurement of the b2 zymes in clinical isolatesofdcinetobacter calcoaceticus. deletion in coliphage lambda. Gene 2, 289-293. Curr. Microbial. 4, 36 l-364. GOLDSTEIN,F. W., BOISIVON,A., LECLERC,P., ACAR, J. BERGOCNE-BEREZIN, E., ZECHOVSKY,N., PIECHAUD,M., F. ( 1977).Sensibilited’hhmophilus sp. aux antibiotiques. VIEU, J. F.. AND BORDINI, A. (1971). Sensibilite aux Transfert de resistancea Escherichia cob. Pathol. Biol. antibiotiques de 240 souches de Moraxella (Acineto 25, 323-332. batter) isoleesd’infections hospitalieres. Evolution sur GOLDSTEIN, F. W., PAPADOPOLJLOU,B., EYQUEM, 3 ans. Pathol. Biol. 19, 2 l-22. M. T., ANDACAR, J. F. (1982). “Progr. Abstr. Intersci. BOUANCHAUD,D. H., SCAVIZZI,M. R., ANDCHABBERT, Conf. Antimicrob. Agents Chemother..” 22nd. Miami, Y. A. (1969). Elimination by ethidium bromide of anFl. Abstract 868. tibiotic resistancein Enterobacteria and staphylococci. GOMEZ-LUS,R., LARRAD,L., RUBI~CALVO, M. C., NAJ. Cm. Microbial. 54, 4 17-425. VARRO,M., AND ASIERRA,M. P. (1980). AAC(3) and CHABBERT,Y. A. ( 1957).Une technique nouvelle d’kude AAC(6’) enzymes produced by R plasmids isolated in de I’action bactericide des associationsd’antibiotiques: general hospital. In “Antibiotic Resistance” (S. Mitle transfert sur cellophane. .4nn. Inst. Pasteur 93, 289sushashi, L. Rosival, and V. Krcmery, eds.), pp. 295299. 303. Springer-Verlag. Berlin, Heidelberg, New York. CHABBERT,Y. A., SCAVIZZI, M. R.. WITCHITZ, J. L., GREY, D., HAMILTON-MILLER,J. M. T.. ANDBRUMFITT, GERBAUD,G. R., AND BOUANCHAUD.D. H. (1972). W. ( 1979). Incidence and mechanisms of resistanceto Incompatibility groups and the classification of Fi- reTmp in clinically isolated Gram-negative bacteria. Chesistance factors. J. Bacferiol. 112, 666-675. motherapy 25, 147-156. GRINDLEY,N. D. F., ANDJOYCE,C. M. (1980). Genetic CLOWES,R. C., AND HAYES,W. (1968). Experiments in and DNA sequenceanalysisof the kanamycin resistance microbial genetics.p. 227. Blackwell, Oxford and EdintransposonTn903. Proc. Nat. Acad. Sci. USA 77,7 176burg. 7180. COURVALIN, P., AND FIANDT, M. ( 1980).Aminoglycosidemodifying enzymes of Staphylococcus aureus: expres- HAAS,M. J., ANDDOWDING,J. E. (1975).Aminoglycosidemodifying enzymes. In “Methods in Enzymology” sion in Escherichia coli. Gene 9, 247-269. (J. H. Hash, ed.), Vol. 43, pp. 61 l-628. Academic Press COLJRVALIN, P., FIANDT, M., ANDDA~IF!&J. ( 1978).DNA New York. relationships between genescoding for aminoglycosidemodifying enzymes from antibiotic producing bacteria HINCHLIFFE, E., AND VIVIAN, A. (1980). Naturally occuring plasmids in Acinetobacter calcoaceticus: a P class and R plasmids. In “Microbiology-1978” (D. SchlesR factor of restricted host range.J. Gen. Microbial. 116, singer, ed.), pp. 262-266. Amer. Sot. Microbial., Wash75-80. ington, D. C.
RESISTANCE PLASMID IN Acinetobacter
147
N. (198 I). Transposable elements in pro- NORMORE, W. N. (1976). Guanine-plus-cytosine (G + C) composition of the DNA of bacteria, fungi, algae karyotes. Annu. Rev. Genet. 15, 341-404. and protozoa. In “Handbook of Biochemistry and MoKONDO, E., HARADA, K., AND MITSUHASHI,S. (1962). lecular Biology” (G. D. Fasman, ed.) pp. 65-240. CRC Drug-resistanceof enteric bacteria. I2-Transduction of Press,Cleveland. the transmissibledrug resistancefactor by bacteriophage NOVICK, R. P., CLOWES,R. C., COHEN,S. N., CURTISS, PlKc. Japan. J. Exp. Med. 32, 139-147. R., III, DAI-TA, N., AND FALKOW,S. (1976). Uniform LABIGNE-ROUSSEL, A., GERBAUD,G.. ANDCOURVALIN, nomenclature for bacterial plasmids: A proposal. ButP. (198 I). Translocation of sequencesencoding antiteriol. Rev. 40, 168-189. biotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez. Mol. Gen. Genet. 182, PATTISHALL,K. H., ACAR, J., BIJRCHALL,J. J., GOLD STEIN,F. W., AND HARVEY, R. J. (1977). Two distinct 390-408. types of Tmpresistant dihydrofolate reductasespecified LABIGNE-ROUSSEL, A., AND~OURVALIN,P. (1983). IS 15, by R. plasmids of different compatibility groups. J Biol. a new insertion sequencewidely spread in R plasmids Chem. 252, 23 19-2323. of gram-negative bacteria. Mol. Gen. Genet. 189, 102112. POURCEL,C., MARCHAL, C., LOUISE,A., FRITSCH,A., ANDTIOLLAIS,P. ( 1979).BacteriophageLambda-E. coh LABIGNE-ROUSSEL, A., WITCHITZ,J. L., AND~OURVALIN, K 12Vector-Host systemfor genecloning and expression P. (1982). Modular evolution of disseminated Inc 7-M under lactose promoter control. izlol. Gen. Genet. 170, plasmids encoding gentamicin resistance. Plusmid 8, KLECKNER,
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LE GOF!=IC, F., ANDMARPEL,A. ( 1974).Methode d’etude des enzymes inactivant les antibiotiques. Etude de la resistancede Moravellu a la tobramycine, la kanamycine et au BBK 8. Nouv. Presse Med. 3(Suppl. 17), 55-56. MANIATIS, T.. JEFFREY,A., AND KLEID, D. G. (1975). Nucleotide sequenceof the rightward operator of phage
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RAHAL, K., GERBAUD,G. R., AND CHABBERT,Y. A. (1973). Caracterisation dun facteur de resistancetransarable de I’ibrio cholerae biotype Eltor. Ann. Microbial. (Inst. Pasteur) 124B, 283-294.
ROBERTS,R. J. (1982). Restriction and modification enzymes and their recognition sequences.Nucleic Acid Res. 10, 117-144. h. Proc. Nat. Acad. Sci. USA 12, I 184-I 188. ROUSSEL, A. F.. ANDCHABBERT,Y. A. (1978). Taxonomy MATTHEW,M., HARRIS,A. M., MARSHALL,M. J., AND and epidemiology of Gram-negative bacterial plasmids Ross, G. W. (1975). The use of analytical isoelectric studied by DNA-DNA filter hybridization in formfocusingfor detection and identification of b-lactamases. amide. J. Gen. Microbial. 104, 269-276. J. Gen. Microbial. 88, 169- 178. SHIMIZU, S.. INOUE, M.. AND MITSUHASHI,S. (1981). MIZON, F. M., GERBALJD,G. R., LECLERC,H., AND Enzymatic adenylylation of spectinomycin by AcineCHABBERT,Y. A. (1978). Presence de plasmides de tohacter calcoaceticus, subsp. anitratus. .I. Antibiot. 34, resistance appartenant au groupe d’incompatibilite C 869-875. chez Aeromonas hydrophita isole d’eaux residuaires. SHINNICK,T. M.. LUND, E.. SMITHIES,O., AND BLATTAnn. Microbial. (Inst. Pasteur) 129B, 19-26. NER, F. R. (1975). Hybridization of labeled RNA to MOROHOSHI,T., AND SAITO,T. (1977). o-lactamase and DNA in agarosegels. Nucleic Acids Res. 2, 1911-1929. &Iactam antibiotics resistancein Acinetobacter anitratus SO,M., GILL, R., ANDFALKOW,S. ( 1975).Thegeneration (syn: A. calcoaceticus). J. Antibiot. 30, 969-973. of a ColE I -Ap cloning vehicle which allows detection MURRAY, B. E., AND MOELLERING,R. C., JR. (1979). of insert DNA. Mol. Gen. Genet. 142, 239-249. Aminoglycoside-modifying enzymes among clinical SOUTHERN,E. M. (1975). Detection of specific sequences isolates of Acinetobacter calcoaceticus subsp. anitratus among DNA fragmentsseparatedby gel electrophoresis. (HerelIeu vuginicola): Explanation for high-level amiJ. Mol. Biol. 98, 503-517. noglycoside resistance.Antimicrob. Agents Chemother. SUMEGI, J.. BREEDVELD,D., HOSSENLOPP,P., AND 15, 190-199. CHAMBON.P. ( 1977).A rapid procedure for purification MURRAY, B. E., AND MOELLER~NG,R. C., JR. (1980). of EcoRI endonuclease.Biochem. Biophys. Res. ComEvidence of plasmid-mediated production of aminomun. 76, 78-85. glycoside modifying enzymes not previously described TOWNER,K. J., AND VIVIAN, A. (1976). RP4-mediated in Acinetobacter. Antimicrob. Agents Chemother. 17, conjugation in Acinetohacter calcoaceticus. J. Gen. Mi30-36.
crohiol. 93, 355-360.
10,
138-147 (1983)
Transferable
Plasmid-Mediated
Antibiotic
Resistance
in Acinetobacter
F. W. GOLDSTEIN,*A. LABIGNE-ROLJSSEL,t*’ G. GERBAUD,? C. CARLIER,~ E. COLLATZ,~” AND P. CouRvALrNt*3 *Laboratoire de Mcrobiologie Mt!dicale, H6pital St. Joseph, 75014 Paris, and TLaboratoire de Biochimie, L..4. CNRS 271. UnitP de Bactkiologie Midicale. Institut Pasteur, F-75724 Paris, France
Received March 16. 1983 Acinetobacter calcoaceticus strain BM2500 was resistant to ampicillin. aminoglycoside-aminocyclitols, chloramphenicol, sulfonamides, and high levels of trimethoprim. Resistanceto ampicillin was due to the presenceof a /3-lactamase(TEM-I) and the aminoglycoside-aminocyclitol resistancewas mediated by phosphotransferase(APH(3’)(5”)1)and adenylyltransferase(AAD(3X9)) activities. The resistance genes were carried by a 167 kilobase plasmid, pIPlO I, belonging to incompatibility group 6-C; the plasmid was self-transferable, at extremely low frequency, to Escherichia co/i by conjugation. Plasmid pIPlO 1 DNA wasanalyzed by agarosegel electrophoresis following restriction endonucleasedigestion, by nucleic acid hybridization, and by CsCl analytical density gradient ultracentrifugation. The results support the hypothesis that plasmid PIP 1031 may have been acquired recently by strain BM2500.
Acinetobacter is a ubiquitous Gram-negative, aerobic, nonfermentative coccobacillus increasingly responsible for nosocomial infections and resistant to antibiotics, in particular penicillins, aminoglycoside-aminocyclitols, chloramphenicol, and tetracyclines (Bergogne-Birezin et al., 197 1; Murray and Moellering, 1979). Acinetobacter is naturally resistant to cephalosporins due to the production of a cephalosporinase (Morohoshi and Saito, 1977) and to low levels of trimethoprim (with minimal inhibitory concentrations (MICs) of 16 to 32 &ml). In certain strains, resistance to penicillins (Devaud et al., 1982), aminoglycosideaminocyclitols (Gomez-Lus et al., 1980; Le Goffic and Martel, 1974; Dowding, 1979; Murray and Moellering, 1979, 1980; Bergogne-Birezin et al., 1980; Shimizu et al., 1981; Devaud et al., 1982) and chloramphenicol (Devaud et al., 1982) is due to en’ Presentaddress:Department of Medical Microbiology. Stanford University School of Medicine. Stanford, Calif. 94305. Z Present address:Laboratoire de Microbiologic, UER 82 Les Cordeliers, Universite de Paris VI, 75270 Paris Cedex 06. 3To whom reprint requests should be addressed. 0147-619X/83 $3.00 Copyright @ 1983 by Academic Press. Inc. All nghls of reproduction in any form re%rVed.
zymes which modify these antibiotics. Suggestive evidence of plasmid-mediated resistance toward penicillins, aminoglycosides, tetracyclines, sulfonamides, and mercury has recently been reported (Murray and Moellering, 1980) and transfer of sulfonamide resistance has been obtained between strains of A. calcoaceticus (Hinchliffe and Vivian, 1980). Resistance (R) plasmid transfer from a clinical isolate of A. calcoaceticusto Escherichia coli has been reported (Gomez-Lus et al., 1980). In this study we describe a strain of A. calcoaceticus subsp. anitratus resistant to penicillins, aminoglycoside-aminocyclitols, chloramphenicol, sulfonamides, and high levels ( 1 mg/ml, or higher) of trimethoprim. The resistancegenes(R determinants) are carried by a plasmid, belonging to incompatibility (Inc) group 6-C. The mechanisms of resistance toward P-lactams and aminoglycoside-amino4 Abbreviations used: ColE I, colicinogenic factor E I ; kb, kilohasc; R plasmid. antibiotic resistanceplasmid; R determinant, antibiotic resistancemediating gene: cRNA. complementary RNA: SIOO,supematant (100,OOOg)of a bacterial extract after sonication; genotypes include aad(33(9). aminoglycoside-aminocyclitol adenylyltransferase3”. 9; aph(3’)(5”), aminoglycoside phosphotransferase Y.5”.
138
139
RESISTANCE PLASMID IN Acinefobacfer
cyclitols are described and the plasmid R determinants are compared with those of known transposons. An exogenous origin for the R plasmid is proposed. MATERIALS
AND METHODS
Bacterial strains. The sourcesand properties of the bacterial strains are listed in Table 1. Media. Tryptic soy broth and agar (Difco) were used. Disc sensitivity tests were done on Mueller-Hinton agar. All incubations were at 37°C. Genetic techniques.Conjugation (Chabbert, 1957) transformation (Davis et al., 1980), determination of incompatibility (Chabbert et al., 1972) and curing of antibiotic resistance traits (Bouanchaud et al., 1969) were performed as described. Assa)?.for aminoglycoside-aminocyclitolmodifying enzymes. The enzymes were assayedas described (Haas and Dowding, 1975).
Preparation ofDNA. High-molecular weight (Labigne-Roussel et al., 1981) and low-molecular weight (Davis et al., 1980) plasmid DNA was purified as described. DNA from bacteriophage Xc1857and derivatives (Courvalin and Fiandt, 1980) and A. calcoaceticus high-molecular weight total DNA (Roussel and Chabbert, 1978) were prepared as described. Agarose gel electrophoresis. DNA was digested with restriction endonucleases in 100 mM Tris-HCl (pH 7.8) 100 tYtM MgC12 for EcoRI and in 6 tnM Tris-HCl (pH 7.0). 6 mM MgC12, 50 mM for HindIII. Agarose gel electrophoresis was as described by Shinnick et al. (1975). Hybridization. 32P-labeledDNA (Maniatis et al., 1975)and complementary RNA (cRNA: Courvalin and Fiandt, 1980) were prepared as described. Restriction endonuclease-generated DNA fragments fractionated by agarose
TABLE I PROPERTIES OF THE BACTERIAL
Number
STRAINS USED
Relevant characteristics and plasmid content’
Strain
Source or reference
BM15
E. coli K12
azi rpoB pro met
Spontaneous mutant of 553 (Clowes and Hayes, 1968)
LA101
E. coli K12
str.4 hsdR- sulI sulll
Derived from C600 (Pourcel et al.. 1979)
BM694
E. coli C
nal.4
Spontaneous mutant of Cla (Labigne-Roussel et al.. 1981)
BM2500
A. calcoacericus
pIPlO Tra+ Inc 6-C Ap Cm Km Sm Su Tpb pIP1032, pIP1033, pIPlO34, pIP1035,pIP1036
Wild strain
BM2500- 1
A. calcoaceticus
pIP1032, pIP1033, pIP1034. pIP1035. pIPlO
Curing of BM2500
BM250 I
E. co/i K12
azi rpoB pro met
Conjugation BM2500 X BM I5
PIP 103I Tra+ Inc 6-C Ap Cm Km Sm Su Tp BM2502
E. co/i K12
str.4 hsdR- sull &II
Conjugation BM2501 X LA101
pIPlO Tra+ Inc 6-C Ap Cm Km Sm Su Tp BM2503
E. coli C
nal.4
pIPlO Tra+ Inc 6-C Ap Cm Km Sm Su Tp ’ Genetic symbols are according to Bachmann et al. (1976) and Novick ’ Designation of plasmid and its phenotypic characters are aligned.
Transformation PIP 103I X BM694
et a/. (I 976).
140
GOLDSTEIN ET AL.
gel electrophoresiswere transferred to cellulose nitrate sheets (Sartorius) as described by Southern (1975). Isoelectric.focusing
in polyacrylamide
gels.
Isoelectric focusing of S100 preparations was as described (Matthew et al.. 1975). DNA base composition. DNA base composition was determined after buoyant density measurements as described (Courvalin et al.. 1974). Enzymes. Restriction endonuclease EcoRI (Siimegi et al., 1977)was purified as described.
Endonuclease Hind111was kindly provided by J. Gardner and R. Jorgensen. Endonuclease BarnHI, from Boehringer, was usedaccording to the manufacturer’s recommendation. Enzyme sources were proteinase K (Merck), lysozyme (Sigma), ribonuclease A (bovine pancreas) (Calbiochem), and E. coli K12 RNA polymerase holoenzyme (Miles). Chemicals. [ l-‘4C]acetyl-coenzyme A ([ “C]CoASAc), adenosine 5’-[a-32P]triphosphate, triethylammonium salt ([a-32P]ATP), adenosine 5’-[y-3’P]triphosphate, triethylammonium salt ([T-~‘P]ATP), and [U“C]adenosine 5’-triphosphate, ammonium salt ( [U-14C]ATP) were obtained from the Radiochemical Centre, Amersham, England. The antibiotics were provided by the following laboratories: gentamicins (Gen) A and B, Schering; Kanamycins (Kan) A, B, C, and amikacin (Ami), Bristol; neomycin B (NeoB), spectinomycin (Spc), Upjohn; paromomycin (Par) and butirosin (But), Parke-Davis; tobramycin (Tob). Lilly: lividomycin (Liv) A, Kowa; ribostamycine (Rib), Meiji: streptomycin (Str), Pfizer; chloramphenicol, Roussel; rifampin. Lepetit; nalidixic acid, Winthrop: trimethoprim, Roche. Sarkosyl (sodium lauryl sarcosinate) was provided by Colgate-Palmolive.
methoprim (Tp) (Table 1). In curing experiments these characters were lost en bloc (approximately 0.3% of 300 colonies tested) and one cured strain, BM2500- 1, was studied further. The genes conferring resistance to ApCmKmSmSuTp were transferred with a very low frequency, about 5 X lo- lo, from BM2500 to E. cofi BM 15 by conjugation (Table 2). These R determinants were further transferred from one transconjugant (strain BM2501, Table 1) to E. cofi LA 101 by conjugation. Selection for transfer of any of these resistance characters (Table 2) revealed cotransfer of all R determinants (in 10 clones studied). One strain, BM2502 (Table l), was selected for further studies. In this case, the frequencies of transfer were 3 to 8 X 10m6 (Table 2). Incompatibility testing of the transconjugant BM2501 revealed that all the acquired characters were borne by a single plasmid belonging to the incompatibility (Inc) group 6C (Chabbert et al., 1972; Datta, 1975). Plasmid Content of BM2500, Its Derivative BM2500-1. and Transconjugant BM2501
The plasmid DNA from strains BM2500, BM2500- 1, and BM250 1 was purified by ultracentrifugation and analyzed by agarosegel electrophoresis, before (Fig. 1, Table 1) and after (data not shown) digestion with EcoRI endonuclease. Comparative analysis of the phenotypes with the plasmid content and the EcoRI-genTABLE 2 FREQUENCYOF PLASMIDTRANSFER
Donor
Recipient
Selected resistance from donor
RESULTS
BM2500
BM15
TP
Plasmid-Mediated Characters Expressed bJ A. calcoaceticus BM2500
BM250 I
LA101
SP Cm Km TP
Frequency” 5 x IO-‘” 8x 3.2 x 5x 7.3 x
Io-c 1o-6 1o-6 1o-6
Acinetobacter calcoaceticus BM2500 encodes resistance to ampicillin (Ap), chlora Frequenciesare expressedrelative to 2 X 10m9bacterial amphenicol (Cm), kanamycin (Km), strep- donors after mating on cellophane membranes (Chabbert, tomycin (Sm), sulfonamide (Su), and tri- 1957) for 18 h.
RESISTANCE BM 2500-l
BM 2500
PLASMID
BM 2501
-11 6.3 7.6
e 6.3
5.5
e 5.4
4.6 I
4.4
FIG. 1. Analysis of plasmid DNA. Plasmid DNA was fractionated by electrophoresis in a 0.6% agarose gel ( 18 X 13 X 0.4 cm) for 18 h at 3 V/cm. Right, plasmids pBR322, 4.4 kb, pBR325, 5.4 kb; ColEI, 6.3 kb; and pSF2124, I 1 kb, were used as molecular size standards. Left, the size of the plasmids is expressed in kilobases. Plasmids pIPlO (upper) and pIPlO (lower) are indicated by white arrows.
141
IN Acinetobacter
erated fragment patterns of plasmid DNA in the individual strains led us to conclude that the wild-type strain BM2500 harbors six plasmids (Table 1). Plasmid pIPlO encoded all the transferable resistances;it had a molecular size of approximately 167 kilobases (kb) and eight EcoRI-generated DNA fragments which were numbered in order of decreasing size. The other plasmids were cryptic. Plasmids pIP1032, pIP1033, pIP1034, pIP1035, and PIP 1036 had molecular sizesof about 70, 8.3, 7.6, 5.5, and 4.6 kb, respectively. Strain BM2500-1 was susceptible to ApCmKmSmSuTp and had lost pIPlO 1. E. cofi strain BM2501 was resistant to ApCmKmSmSuTp after acquisition of PIP 1031. Plasmid pIPlO was detected in BM2502, after conjugation, and in BM2503, after transformation (Table
1).
Mechanisms of Plasmid-Mediated Resistance to Antibiotics in BM2500 Strain BM2500, its derivative BM2500- 1, and the transconjugant BM2501 (Table 1)
A.calcoaceticus E. ___coli __- - . 812501
112500
_---. 1 FIG. 2. Substrate profiles of enzymes extracted from A. calcoaceficusstrain BM2500 (solid lines) and E. (plain area) or adenylylation (hatched area) are expressed relative to neomycin B or streptomycin as 10096, respectively. No acetylation of neomycin B, streptomycin, or spectinomycin, no phosphorylation of streptomycin or spectinomycin, and no adenylylation of neomycin B were detected in these strains. No aminoglycoside-modifying activity was detected in the susceptible strain BM2500- 1.
coli strain BM250 I (dashed lines). Phosphorylation
142
GOLDSTEIN ET AL.
were examined for aminoglycoside-aminocyclitol-modifying activities. Strains harboring plasmid PIP 103I were found to contain aminoglycoside phosphotransferase (APH) and aminoglycoside-aminocyclitol adenylyltransferase(AAD) but no acetyltransferase (AAC) activities. The substrateprofiles of the enzymes extracted from strains BM2500 and BM250 1 are shown in Fig. 2. The fact that kanamycin B was phosphorylated and tobramycin not, indicates that the 3’-hydroxyl group is the site of phosphorylation (APH(3’)). That lividomycin A is a substrate for phosphorylation but butirosin not, indicates that the enzyme is of type I (APH(3’)(5”)1) (Davies and Smith, 1978). The adenylylating enzyme is equally effective against streptomycin and spectinomycin, thus probably an (AAD(3”)(9)) (Davies and Smith, 1978). Strain BM2500 is resistant to penicillins and cephalosporins. Escherichia coli BM250 I is resistant to penicillins but not to cephalospotins and A. calcoaceticusBM2500- I is resistant solely to cephalosporins. Strain BM2500 encodestwo P-lactamaseswith isoelectric points of about 9.5 and 5.4 (Fig. 3). The former enzyme, also present in BM2500- 1, should correspond to the chromosome-mediated cephalosporinase (Morohoshi and Saito, 1977). The latter enzyme, also detected in BM2501 is a TEM- 1 penicillinase encoded by PIP 1031. We did not find any inactivation of chloramphenicol by strains BM2500 and BM250 I using a microbiological detection technique (Goldstein et al., 1977). Analysis of DNA bv Hybridization Total DNA from BM2500 and pIPlO DNA were hybridized to plasmid or bacteriophage probes (Fig. 4). The properties of the template DNAs used to localize the R determinants and to search for the presence of insertion sequences(IS) are summarized in Table 3. In each experiment, the reaction with the pIPlO 1 probe was used as a control. No differences were observed between the hybridization patterns of PIP 103I (Fig. 4B) and BM2500 total DNA (Fig. 4C). The kanamycin R determinant was localized with X::Tn601 and X probes on EcoRl
.w
-C
,:!:i ‘,,I
_ 5.6 - 5.4
-----_ FIG.3. Analytical isoelectricfocusing.&lactamases from the strains indicated on the top were focusedand revealed by nitrocefm (Glaxo). 1, E. coii K12/pSF2124 encoding a TEM-I &lactamase (PI = 5.4): 2, E. co/i K12/RP4 encoding a TEM-2 j3-lactamase(PI = 5.6): C. cephalosporinase.
fragment 4 of pIP 103I. Transposableelements Tn6 (Berg et al., 1975) and Tn601 (Davies et al., 1977) later designated Tn903 (Table 3) confer resistance to kanamycin by synthesis of APH(3’)(5”)-I enzymes with substrate profiles similar to that encoded by pIPlO (Fig. 2). The phosphotransferase genes in the two transposons are related (Courvalin et al., 1979). Both Tn6 and Tn602 are composite classI elements (Kleckner, 1981) in which the region encoding kanamycin resistance is flanked by insertion sequences(IS), IS903 in the caseof Tn601 (Grindley and Joyce, 1980) and IS15 (Labigne-Roussel and Courvalin, 1983) in the case of Tn6. Both X::Tn6 and X: :Tn60Z hybridized to EcoRI fragment 4 of pIPlO 1 and X: :Tn6 hybridized, in addition, to fragment 3. Hybridization experiments (Labigne-Roussel and Courvalin, 1983) re-
143
RESISTANCE PLASMID IN Acinetobacler
PROBES
,
plP1031
(*I
(6)
BM2500
Total
DNA
‘j
DRIVERS (EcoRI)
@I
FIG. 4. Analysis of DNA by hybridization. Plasmid pIPlO I DNA or BM2500 total DNA (drivers), were digested with EcuRI. The resulting fragments were fractionated by gel electrophoresis (0.7% agarose, 18 X 13 X 0.4 cm) for 12 h at 3 V/cm, transferred to nitrocellulose sheets, and hybridized to in vitro 32Plabeled DNA (or cRNA for ColE 1::Tn7*) (probes). The size of the eight PIP 1031 EcoRI-generated DNA fragments (A) was determined using Xc1857 DNA, 48 kb, digested with EcoRI, SmnI, and EcoRI + Hind111 as molecular size standard. Bands 5 to 8 in (C), although faint on the photograph were clearly visible on the autoradiogram.
vealed the presence of a natural deletion derivative of ISIS, ISI5A, in pIPlO EcoRI fragments 3 and 4. Plasmid PIP 1031 encodes a TEM- 1 fl-lactamase (Fig. 3). The pSF2124 (Ap) probe hybridized to fragment 3 and, although with a weaker intensity, also to fragment 4 of PIP 1031. The ubiquitous Tn3 element does not contain an EcoRI site. In pIPlO 1, we cannot discriminate between the presence of two ampicillin R determinants or the occurrence of an EcoRI site in its P-lactamasegene. The EcoRI (SmSu) insert in pCH 13 allowed us to assignthe streptomycin and sulfonamide determinants to EcoRI fragments 3 and 6 of pIPlO 1. As in the case of ampicillin resistance, we do not know if there is an EcoRI site in the Sm or Su R determinants of pIPlO 1 or if there is a duplication of either or both genes. A TEM-1 gene possessingan
EcoRI site and a plasmid carrying two related AAD(3”)(9) genes have been described (Iabigne-Roussel et al., 1982). The transposable element Tn7 confers resistance to trimethoprim by synthesis of a dihydrofolate reductase type I (Pattishall et al., 1977) and to streptomycin by synthesis of an AAD(3”)(9). We detected homology between ColE 1: :Tn7 DNA labeled by nick-translation and pIPlO 1 EcoRI fragments 3 and 6 but not between a ColE 1: :Tn 7 cRNA probe and pIPlO 1. While the dfr1 gene is transcribed under the conditions used, the aad gene is not (Iabigne-Roussel et al., 1982). These results agreewith the existence, on pIPlO 1, of either two homologous Sm R determinants or the occurrence of an EcoRI site in the region encoding the adenylylating enzyme; in addition, they indicate that resistance to trimethoprim in BM2500 is not due to a dihydrofolate re-
144
GOLDSTEIN ET AL. TABLE 3 PLASMIDS
AND BACTERIOPHAGES
Plasmid or bacteriophage ColEl pSF2I24 pCH13’ ColE I : :Tn7 xc X::Tn6d X::Tn601’ X::ISI’ PlCm
USED AS PROBES FOR HYBRIDIZATION
Relevant genotype”
Source or reference
-
Bazaral and Helinski (1972) So et al. ( 1975) J. Davies N. Datta Fiandt ef al. (I 977) Berg er al. (I 975) Davies et al. (I 977) P. Starlinger Kondo et al. ( 1964)
bla (TEM- 1)
ad (3”)(9), sul aad (Y)(9). dfr1 aph (3’)(Y)-1 uph (3’)(5”)-I
cat
’ Genetic symbols are according to Novick et al. ( 1976) and Davies and Smith ( 1978). bpCH13, ColElQ {r&(3”)(9), sul). ‘A, XcI857. d X::Tn6, hb5 1565I9 ~I857 Sam7::Tn6. kindly provided by R. Jorgensen. ‘X::Tn60l, Xb515b519 c1857 .Sam7::Tn601.
’ h::ISI, XI, Xdgur ~1857S~68-0p306::ISI, kindly provided by P. Starlinger.
ductase closely related in sequence to that of Tn7. No hybridization was detected between pIPlO and Xc1857,ColEl, X::ISZ, or PlCm. The latter is consistent with our failure to detect inactivation of chloramphenicol by strains BM2500 and BM2501.
icol, kanamycin, streptomycin, sulfonamides, and trimethoprim in A. calcoaceticus subsp. anitratus strain BM2500 are located on a 167 A I
Base Composition of DNA Chromosomal and plasmid DNA from BM2500 were analyzed by CsCl analytical density gradient ultracentrifugation (Fig. 5). The buoyant density of the chromosomal DNA was 1.6907, corresponding to a G + C content of 37.5%. The buoyant density of pIPlO 1 was 1.700 which corresponds to 46.5%G + C. When plasmid DNA from strain BM2500- 1 was analyzed, a peak and a shoulder were seen. The peak corresponding to a density of 1.693 (39.5% G + C) should represent PIP 1032; the shoulder, corresponding to a density of 1.69 (G + C 36.5%) should represent the pool of the small molecular size plasmids pIP1033, pIPlO34, pIP1035, and pIP1036. DISCUSSION We have demonstrated that the genesconferring resistanceto ampicillin, chloramphen-
:’
---#I---
PIP
1031
1 I -
-J
BY 2500.1 ptm8mld DNA
ii ---
-.J
BM 2500 total DNA 1.703
1.700
1.6907
FIG.5. Analysis of DNA by isopycnic ultracentriiirgation in CsCI. Bacteriophage Xc1857DNA (p = 1.703 g/cm-‘) was included as an internal standard.
RESISTANCE PLASMID IN Acinetobacter
kb plasmid designated pIPlO 1. The loss or acquisition of this resistancephenotype is correlated with the absenceor presenceof plasmid pIPlO DNA (Fig. 1). In strain BM2500 resistance to streptomycin and spectinomycin is mediated by a 3” 9-adenylyltransferase (AAD(3”)(9)) and resistanceto kanamycin and structurally related compounds is due to the synthesis of a 3’5” phosphotransferase of type I (APH( 3’)(S’)-I) (Fig. 2). Enzymes with identical site specificity have been described in Acinetobacter (Bergogne-B&-&n et al., 1980; Gomez-Luz, 1980; Murray and Moellering, 1980; Shimizu et al., 1981; Devaud et al.. 1982). Resistanceto ampicillin, carboxy- and ureidopenicillins is due to the presenceof a TEM- 1 /I-lactamase (Figs. 3 and 4). Resistance of strain BM2500 to chloramphenicol does not appear to take place by inactivation of the antibiotic; resistance to sulfonamide is also by an unknown mechanism. Resistance to trimethoprim is novel in Acinetobacter but the biochemical mechanism for this resistance remains unknown. As inferred from hybridization experiments (Fig. 4) it is not due to a dihydrofolate reductase of type I. Incidentally. a dihydrofolate reductase of type II has been found associatedwith an Inc 6-C plasmid (seebelow) isolated in the same hospital as pIPlO 1 (Pattishall et al., 1977). The G + C content of BM2500 chromosomal DNA is 37.5% (Fig. 5) a ratio expected for a member of this species(Normore, 1976). This value, which represents an average for the whole genome, is rather close to those (39.5 and 36.5%) estimated for the cryptic plasmids of this strain. However, the base composition of PIP 1031 is 46.5% G + C which presumably indicates a foreign origin of this replicon. Plasmid pIPlO 1 is self-transferable by conjugation, at extremely low frequency (Table 2) to E. co/i. Curiously, this transfer could only be obtained with the initial isolate of A. calcoaceticus BM2500. As judged from the EcoRI fragment pattern of BM2500 plasmid DNA, and from hybridization between the total DNA of initial or subsequent cultures of this strain and a PIP 1031 probe (Fig. 4) the
145
loss of this property was not due to major rearrangements in the plasmid genome nor to integration of the R determinants into the host chromosome. This phenomenon leads us to speculate that plasmid PIP 1031 was acquired only extremely recently by strain BM2500. This is in contrast with the situation in E. coli where pIPlO 1 is stable and can be easily transferred by conjugation (Table 2) or transformation (Table 1). Plasmid pIPlO belongs to the incompatibility group 6-C (Chabbert et al., 1972). Like Inc P, and to a lesser extent Inc 7-M plasmids, plasmids of this group are ubiquitous. They are frequently encountered in France (J. Witchitz, personal communication) especially in Enterobacteriaceae (Acar et al., 1977). They also possessa wide host range and have been found in many diverse pathogenic bacterial species including rleromonas hydrophila (Mizon et al., 1978), Pseudomonas aeruginosa (Chabbert et al., 1972) and Vibrio choferue (Rahal et al., 1973). This broad host range may be related to the fact that the DNA of the Inc 6-C plasmids is relatively inert to restriction endonuclease digestion. For example, EcoRI (Fig. 4) HindIII, BumHI, and Pst 1 (J. Witchitz, personal communication and our unpublished data) generateonly a few large DNA fragments. Becausethese plasmids are extremely common and can easily cross generic barriers, the Inc 6-C plasmids are likely vectors for the introduction of new genetic information to genera like Acinetobacter, which is known to produce a large variety of restriction endonucleases(Roberts, 1982).The same seemsto be true for the Inc P plasmids which have been found to transfer resistance genes from clinical isolates of Acinetobacter to E. cofi (Gomez-Lus et al., 1980) or from E. coli to .kinetobacter (Towner and Vivian, 1976). The -4. calcoaceticus strain BM2500 was isolated from a clinical specimen at the Saint Joseph Hospital in Paris. In this ecosystem, the ApCmKmSmSuTp determinants are commonplace (F. Goldstein, unpublished observation) and are most frequently (i.e., in about 40% of the cases)borne by Inc 6-C plasmids (Acar et al., 1977;Goldstein et al.. 1982).
146
GOLDSTEIN
ET AL.
We then consider the presence of plasmid COURVALIN,P. M., CARLIER,C., CROISSANT,0.. AND BLANGY,D. (1974). Identification of two plasmids dePIP 1031 in a wild strain of Acinetobacter as termining resistanceto tetracycline and to erythromycin an indication that C type plasmids have an in group D Streptococcus. Mol. Gen. Genet. 132, 18 I even broader host range than was suspected. 182. DATTA, N. (1975). Epidemiology and classification of plasmids.In “Microbiology-1974” (D. Schlessinger.ed.). ACKNOWLEDGMENTS pp. 9-15. Amer. Sot. Microbial. Washington, D. C. We thank A. Fritsch for buoyant density determinations, DAVIES, J., BERG, D., JORGENSEN,R., FIANDT, M.. 0. Rouelland for secretarialassistance,and Y. A. Chabbert HUANG, T. S. R.. COURVALIN,P., AND SEHLOR‘,J. for material support and otherwise. (1977). Transposable neomycin phosphotransferases.In “R-factors: Their Properties and Possible Control” (J. REFERENCES Drews and G. Hogenauer, eds.) pp. 10I - I IO. SpringerACAR, J. 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