a Service de BactériologieVirologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre Cedex; b Service de Microbiologie, Centre Hospitalier de Troyes, Troyes, France
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Some ß-lactamase genes may be encoded in gene cassettes that are present in the variable region of integrons.16 Gene cassettes are discrete mobile units comprising a gene, usually an antibiotic resistance gene, and a recombination site that is recognized by an integrase.1618 The cassette-associated recombination sites, known as 59 base elements, are located downstream of inserted genes and are of variable length.1720 Class 1 integrons, which are isolated predominantly from antibiotic-resistant clinical isolates, commonly possess two conserved regions located on either side of the integrated gene cassettes.18 The 5'-conserved segment (5'-CS) includes a gene, intI1, encoding the integrase; attI1, the cassette integration site; and the promoter, Pc (formerly Pant), that is responsible for the expression of inserted cassette genes.18 The 3'-conserved segment (3'-CS) includes, along with another open reading frame (orf5), a disinfectant-resistance determinant (qacEDelta;1) and a sulphonamide-resistance determinant (sul1).18 Primers to these conserved segments are usually used for the amplification of the variable regions of class 1 integrons. In addition, defining a cassette when it is the only integrated cassette is done normally by identifying the known boundaries of the 5' and 3'-CSs. While most class D ESBL genes are found on integrons,21 the only class A ESBL genes present on the variable region of integrons are blaVEB-113 and the blaGES-1 reported recently from K. pneumoniae ORI-1.22
In this report, we have analysed the ß-lactamase gene content of a P. mirabilis clinical isolate for which synergy between ceftazidime and clavulanic acid was found along with unusual synergy between cefoxitin and cefuroxime in a disc diffusion assay. We have compared the integron, In55, identified in P. mirabilis Lil-1 with those of E. coli MG-1,13 P. aeruginosa JES and P. aeruginosa PaTh2,14,15 the latter two being from Thailand.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
P. mirabilis Lil-1 and K. pneumoniae Lil-2 were identified with the API-20E system (bioMérieux, Marcy-l'Etoile, France). Electrocompetent E. coli DH10B (Gibco-BRL Life Technologies, Eragny, France) was used as the recipient in electroporation experiments and E. coli JM109, which is naturally resistant to nalidixic acid, was used for the conjugation experiments.23 E. coli MG-1 has been described previously.13 E. coli NCTC 50192 harbouring 154, 66, 38 and 7 kb plasmids was used as a plasmid-containing reference strain.24
Routine antibiograms were determined by the disc diffusion method on MuellerHinton agar (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France). The antimicrobial agents and their sources have been described elsewhere.25 MICs of selected ß-lactams were determined by an agar dilution technique on MuellerHinton plates with a Steers multiple inoculator and an inoculum of 1 x 104 cfu/spot.25 All plates were incubated at 37°C for 18 h. MICs of ß- lactams were determined alone or in combination with a fixed concentration of clavulanic acid (2 mg/L) or tazobactam (4 mg/L). MIC results were interpreted according to NCCLS guidelines.26
Plasmid content, mating-out assays and electroporation
Plasmid DNA of P. mirabilis Lil-1 was prepared using a Qiagen plasmid DNA maxi kit according to the manufacturer's instructions (Qiagen, Courtaboeuf, France) and according to Kieser's plasmid extraction protocol.27 The P. mirabilis Lil-1 plasmid DNA was analysed by electrophoresis on a 0.8% agarose gel, containing ethidium bromide 0.15 mg/L and was compared with that isolated previously from E. coli MG-1 containing blaVEB-1 and with those extracted from E. coli NCTC 50192.24
The extracted plasmid DNA from P. mirabilis Lil-1 was transferred into E. coli DH10B by electroporation using a Gene Pulser II according to the manufacturer's instructions (Bio-Rad, Ivry/Seine, France). Recombinant bacteria were plated on to Trypticase Soy (TS) agar plates containing amoxycillin 100 mg/L. The plasmids were again extracted using a Qiagen maxi column kit and the sizes were estimated by restriction endonuclease digestion (Amersham Pharmacia Biotech, Orsay, France).
Direct transfer of the ß-lactam resistance phenotype from P. mirabilis Lil-1 into nalidixic acid-resistant E. coli JM109 was attempted by liquid and solid mating-out assays at 37°C.25 Transconjugants were selected on TS agar plates containing nalidixic acid 100 mg/L and amoxycillin 100 mg/L.
Isoelectric focusing
ß-Lactamase extracts were prepared as described13 and were subjected to analytical isoelectric focusing (IEF) on a pH 3.59.5 ampholine polyacrylamide gel (Amersham Pharmacia Biotech) for 3 h at 10 W constant power on a flatbed apparatus (FBE-3000, Amersham Pharmacia Biotech). The ß-lactamases were visualized with an overlay of nitrocefin 0.2 g/L in 0.1 M phosphate buffer pH 7.0. The pIs of the ß-lactamases extracted from P. mirabilis Lil-1 and from the E. coli JM109(pLil-1) transconjugant were determined by comparison with those of known ß-lactamases, including VEB-1, TEM-1 and OXA-10 extracted from E. coli MG-1.13
PCR analyses and sequencing
Standard PCR experiments were performed as described.14,28 The PCR amplification and the primers (TEM-F, TEM-B, SHV-F, SHV-B, PER-1/2F, PER-1/2B, TOHO-1F, TOHO-2B, OXA-10-A, OXA10-B, CTXM-F and CTXM-B) used to search for known ß-lactamase genes (blaTEM, blaSHV, blaPER-1/2, blaTOHO-1/2 and blaCTX-M-1/6) in P. mirabilis Lil-1 and K. pneumoniae Lil-2 have been described previously.22 For PCR mapping of the integron, 500 ng total DNA from P. mirabilis Lil-1 and from E. coli MG-1 were used in standard PCR mixtures.14 All the PCR amplifications were performed using previously described PCR primers14,25 and those shown in Table 1 and in Figure 1, lane A, with the following amplification programme: 10 min, 94°C; 35 cycles of 1 min at 94°C, 1 min at 55°C, 3 min at 72°C; followed by a final extension of 10 min at 72°C. In some cases, long-range PCR conditions were used in order to increase the yield of the large PCR products (Long Range PCR kit, PerkinElmer, Les Ullis, France). VEB-F and VEB-B internal primers for blaVEB-1 were used for the detection of the blaVEB-1 gene, while VEBcas-F and VEBcas-B, located at each end of the blaVEB-1 cassette, were used to amplify the entire blaVEB-1 gene cassette.10 The AACA1 and AADB primers were used to study the immediate genetic environment of blaVEB-1. The 5'- and 3'-CS primers were used in combination with VEBINV1 and VEBINV2, respectively, both primers reading outwards from blaVEB-1, in order to determine the size of the variable region of the integron.14 INT1-B, INT2-B and INT3-B, intragenic primers specific for the three integrase gene types (1, 2 and 3),29 together with VEBINV1 primers were used to demonstrate the collinearity of the integrase with blaVEB-1, while primers QACE
1-B, SUL1-B and ORF5-B together with VEBINV2 were used to analyse the genetic content of the 3'-CS.10 INT-FLANK, a primer that hybridizes to the 25 bp inverted repeat IRi and IRt sequences found at the boundaries of several class 1 integrons,30 and 5'-CSINV, a primer hybridizing to the complementary sequence of the 5'-CS primer, were used to amplify a conserved flanking sequence of the class 1 integrase gene.
|
Restriction digestion and hybridization experiments
HindIII- and BamHI-digested whole-cell DNA of P. mirabilis Lil-1, E. coli MG-1 and their electrotransformants were analysed on a 0.7% agarose gel. After electrophoresis, the gels were stained in ethidium bromide 0.5 mg/L and the DNA fragments were transferred on to Hybond N+ membrane (Amersham Pharmacia Biotech) using a vacuum blotting system (Amersham Pharmacia Biotech). Southern hybridizations were performed using an enhanced chemiluminescence (ECL) non-radioactive kit as described by the manufacturer (Amersham Pharmacia Biotech). The probe consisted of a 650 bp intragenic blaVEB-1 PCR fragment amplified from plasmid pRLT-1,13 a recombinant plasmid containing the entire blaVEB-1 gene.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
P. mirabilis Lil-1 was isolated in July 1999, at the hôpital Antoine Béclère, Clamart (a suburb of Paris), France, from the pus of a 2 month old Vietnamese boy adopted by a French family. The child had previously been hospitalized at the Saigon General Hospital in Vietnam, where he was treated for diarrhoea and dehydration. Before his arrival in France, he had a history of scabies and he developed erythematous and maculopapular lesions, which became vesicular and pustular, all over his body. Upon arrival in France, pus samples from his skin lesions were analysed. Four bacterial strains were recovered: a methicillin-resistant strain of Staphylococcus aureus, an ESBL-producing K. pneumoniae Lil-2 isolate, a P. aeruginosa with a wild-type ß-lactam resistance profile and an ESBL-producing P. mirabilis isolate Lil-1 that showed unusual synergy between cefoxitin and cefuroxime on a routine antibiogram, suggestive of an uncommon ESBL. This child probably acquired these bacteria in Vietnam since no other isolate with a similar resistance pattern had been identified in the same hospital during the same time period.
A routine antibiogram revealed that P. mirabilis Lil-1 was resistant to multiple antibiotics, including chloramphenicol, fosfomycin, amikacin, gentamicin, kanamycin, spectinomycin, streptomycin, tobramycin, sulphonamides, tetracycline, trimethoprim and trimethoprimsulphamethoxazole. MICs showed that P. mirabilis Lil-1 was resistant to amino-, carboxy- and ureidopenicillins, and to restricted and extended-spectrum cephalosporins (Table II). This ß-lactam resistance pattern was antagonized by addition of clavulanic acid and tazobactam. MICs of broad-spectrum penicillins for E. coli MG-1 were higher, probably because this isolate expressed, in addition to VEB-1, a TEM-1 ß-lactamase (Figure 1
).13
|
In order to investigate the molecular basis of the ß-lactam resistance observed in P. mirabilis Lil-1, PCR was performed. Negative results were obtained with TEM-, SHV-, PER-1/2-, CTXM-1/6- or TOHO-1/2-specific primers. However, blaVEB-1- and blaOXA-10-specific primers gave PCR products of the expected sizes (650 and 760 bp, respectively; data not shown). PCR amplification followed by direct sequencing of the entire blaVEB-1 gene revealed 100% identity with blaVEB-1 found in E. coli MG-1.13 Similarly, sequencing of the blaOXA-10 PCR product revealed 100% identity with blaOXA-10 from P. aeruginosa POW151.31 These results were confirmed by IEF experiments, which showed that P. mirabilis Lil-1 produced two ß-lactamases, with pIs of 7.4 (which corresponds to the pI of VEB-1 ß-lactamase) and 6.1 (which corresponds to the expected value for OXA-10) (Figure 1). Interestingly, both ß-lactamases were also present in E. coli MG-1 in addition to an enzyme of pI 5.4, characterized previously as TEM-1.13 The level of expression of these ß-lactamases, as deduced from the relative ß-lactamase intensities on the IEF gel, was different in E. coli MG-1 and P. mirabilis Lil-1, which may explain why the MICs of oxyimino-cephalosporins are higher for P. mirabilis Lil-1 than for E. coli MG-1. However, identical ESBL genes usually provide lower MICs for P. mirabilis than for E. coli.3,8 When a second ESBL-producing isolate, K. pneumoniae Lil-2, was isolated from the same patient, PCR experiments with primers specific for ß-lactamases were also performed. Despite several attempts, no positive PCR signal was obtained with blaVEB-1 primers, while blaTEM and blaSHV primers gave positive PCR results. Therefore it is likely that the ESBL present in K. pneumoniae Lil-2 was not a VEB-1-type ß-lactamase. However, the K. pneumoniae Lil-2 ß-lactamases were not characterized further.
PCR mapping of the integron from P. mirabilis Lil-1 and E. coli MG-1
Analysis of the genetic environment of blaVEB-1 revealed key signatures of gene cassettes. The veb1 gene cassette in P. mirabilis Lil-1 was identical to that in E. coli MG-1. The veb1 gene cassette was 1059 bp long and its 59 base element was 133 bp long. Using primers specific for aadB and aacA1, the known immediate genetic environment of blaVEB-1 in E. coli MG-113 (Figure 2b), a 2.7 kb DNA fragment was amplified and subsequently sequenced from both isolates (Figure 2a
). Sequence analysis revealed identical sequences from both strains. The aadB gene cassette, which encodes an aminoglycoside adenyl transferase that confers resistance to gentamicin, kanamycin and tobramycin, was identical to other sequenced aadB gene cassettes.13,32,33 The AacA1orfG fusion cassette was first described as integron-located on an E. coli plasmid, pNR79 (GenBank accession number AF047479). AacA1 codes for an aminoglycoside 6'-N-acetyltransferase which confers resistance to amikacin, tobramycin and netilmicin.33
|
Most of the oxacillinase genes so far identified are located on the variable region of integrons.21 Since blaOXA-10 was identified by PCR and confirmed by IEF, a PCR approach was chosen to see whether it was located on In55 or another integron. Using OXA10-F and 3'-CS primers, a 1.7 kb PCR fragment was generated, indicating the presence of the oxa10 cassette on a class 1 integron. Using VEBINV2 and OXA10-B, a 4.2 kb fragment was amplified, indicating that the two genes are collinear on the same integron.
PCR amplification using type 1 integrase gene- and blaVEB-1-specific primers revealed the presence of an intact type 1 integrase gene in the 5'-CS of P. mirabilis Lil-1. The same PCR remained negative, despite several attempts using primers specific for type 1, but also for type 2 and type 3 integrase genes with E. coli MG-1 DNA as template.29 To test whether the integrase gene in E. coli MG-1 had a deletion of part of the integrase gene or a mutation in the primer-binding site, PCR using INT-FLANK and 5'-CSINV primers was performed. INT-FLANK recognizes the inverted repeat IRi sequence that is conserved and found at the boundaries of most class 1 integrons characterized so far,30 while 5'-CSINV binds to a complementary sequence of the 5'-CS primer-binding site. As expected, a 1.3 kb amplimer was obtained with P. mirabilis Lil-1, while no amplification was observed for the E. coli MG-1 DNA. These data indicate that E. coli MG-1 may carry a different integrase gene, or may have an integrase gene located too far from the 5'-CS to be detected by PCR amplification even under long-range PCR conditions, or may lack an integrase gene. Similarly, PCR amplification using, qacEDelta;1-, sul1-, orf5- and blaVEB-1-specific primers indicated that the qacEDelta;1, sul1 and orf5 genes were present in the 3'-CS of both integrons, typical of sul1-associated integrons.18
Taken together, these results indicate that the variable regions of both integrons containing blaVEB-1 are similar in size but differ at least by the absence of a type 1 integrase gene in E. coli MG-1 and by differences within the integrase flanking sequences. There were major differences between the structures of the P. mirabilis Lil-1 and E. coli MG-1 integrons and those found in the two P. aeruginosa isolates from Thailand (Figure 1, lanes AD). The latter possess either one15 or two14 insertion sequences in their 5'-CS and in both cases, the blaVEB-1 gene cassette is located at the first position in the integron (Figure 1
, lanes C and D). Interestingly, the gene cassette immediately downstream of blaVEB-1, aadB, was identical in the four integrons (Figure 1
, lanes AD). This observation strengthened the hypothesis that these integrons may derive from a common ancestor14 and that they may have evolved according to either geographical or species specificities.
Genetic support of In55
Plasmid DNA preparation from P. mirabilis Lil-1 revealed the presence of three large plasmids of 35, 150 and 190 kb. The largest plasmid, pLil-1, coded for blaVEB-1, as shown by hybridization experiments (data not shown). This plasmid was transferred by electroporation into E. coli DH10B, resulting in the transfer of resistance to chloramphenicol, extended-spectrum ß-lactams, gentamicin, kanamycin, streptomycin, spectinomycin, sulphonamides and tobramycin. These results indicated that blaVEB1 was plasmid-borne. pLil-1 was also transferred by conjugation at low frequency (106) into an E. coli JM109 recipient strain. E. coli JM109 transconjugants harbouring the natural plasmid from P. mirabilis Lil-1 or that from E. coli MG-1 displayed similar ß-lactam MICs, except the ticarcillin MIC, which was twice that for E. coli JM109(pNLT-1) (Table II). This difference may reflect differences in expression or differences in plasmid structure and/or replication. When the associated antibiotic markers were considered, one major difference between bacteria carrying the two plasmids was observed: E. coli JM109(pLil-1) remained susceptible to trimethoprim and trimethoprimsulphamethoxazole, while E. coli JM109(pNLT-1) was resistant (data not shown). Both plasmids encoded resistance to chloramphenicol, gentamicin, kanamycin, spectinomycin, streptomycin, sulphonamides and tobramycin.
pNLT-1 was 180 kb in size, i.e. 10 kb smaller than pLil-1 (data not shown). Thus different plasmids were present in these enterobacterial strains. In addition, BamHI-digested total genomic DNA from both strains and from their electrotransformants, when hybridized with a blaVEB-1- specific probe, revealed two hybridization signals of different size (Figure 3), indicating again that the genetic environment of the two integrons is different, at least in their 5'-CS region. While an expected fragment of 6.5 kb was observed with P. mirabilis Lil-1 (Figures 2a and 3
) a fragment of 10 kb was observed with E. coli MG-1 (Figure 1
, lane B, and Figure 2
). When their genomic DNA was digested with HindIII, an enzyme that cuts within the integron of both isolates, the expected 6.3 kb fragment was observed for both isolates (Figures 2a and b and 3
), underlining the identity of the variable regions of both integrons.
|
|
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Chanal, C., Bonnet, R., De Champs, C., Romaszko, J. P., Sirot, D. & Sirot, J. (1998). Surveillance de la résistance aux ß-lactamases et diversité moléculaire des enzymes de type pénicillinase chez Proteus mirabilis. In Program and Abstracts of the Eighteenth Interdisciplinary Meeting on Anti-Infection Chemotherapy. Société Française de Microbiologie, Paris.
3 . Mariotte, S., Nordmann, P. & Nicolas, M. H. (1994). Extended-spectrum ß-lactamase in Proteus mirabilis. Journal of Antimicrobial Chemotherapy 33, 92535.[Abstract]
4 . Verdet, C., Arlet, G., Ben Redjeb, S., Ben Hassen, A., Lagrange, P. H. & Philippon, A. (1998). Characterisation of CMY-4, an AmpC-type plasmid-mediated ß-lactamase in a Tunisian clinical isolate of Proteus mirabilis. FEMS Microbiological Letters 169, 23540.[ISI][Medline]
5 . Bauernfeind, A., Stemplinger, I., Jungwirth, R., Mangold, P., Amann, S., Akalin, E. et al. (1996). Characterization of ß-lactamase gene blaPER-2, which encodes an extended-spectrum class A ß-lactamase. Antimicrobial Agents and Chemotherapy 40, 61620[Abstract]
6 . Bauernfeind, A., Stemplinger, I., Jungwirth, R., Ernst, S. & Casellas, J. M. (1996). Sequence of ß-lactamase genes encoding CTXM-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequence with those of other ß-lactamases. Antimicrobial Agents and Chemotherapy 40, 50913.[Abstract]
7 . Nordmann, P. (1998). Trends in ß-lactam resistance among Enterobacteriaceae. Clinical Infectious Diseases 27, Suppl. 1, S1006.[ISI][Medline]
8 . Chanal, C., Sirot, D., Romaszko, J. P., Bret, L. & Sirot, J. (1996). Survey of prevalence of extended spectrum ß-lactamases among Enterobacteriaceae. Journal of Antimicrobial Chemotherapy 38, 12732.[Abstract]
9 . Ambler, R. P. (1980). The structure of ß-lactamases. Philosophical Transactions of the Royal Society of London, Series B, Biological Sciences 289, 32131.[ISI][Medline]
10 . Nordmann, P. & Naas, T. (1997). The increasing problem of resistance to cephalosporins. Current Opinion in Infectious Diseases 10, 4359.[ISI]
11
.
Pitout, J. D., Thomson, K. S., Hanson, N. D., Ehrhardt, A. F., Moland, E. S. & Sanders, C. C. (1998). ß-Lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa. Antimicrobial Agents and Chemotherapy 42, 13504.
12
.
Ma, L., Ishii, Y., Ishiguro, M., Matsuzawa, H. & Yamaguchi, Y. (1998). Cloning and sequence of the gene encoding Toho-2, a class A ß-lactamase preferentially inhibited by tazobactam. Antimicrobial Agents and Chemotherapy 42, 11816.
13
.
Poirel, L., Naas, T., Guibert, M., Chaibi, E. B., Labia, R. & Nordmann, P. (1999). Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum ß-lactamase encoded by an Escherichia coli integron gene. Antimicrobial Agents and Chemotherapy 43, 57381.
14 . Naas, T., Poirel, L., Karim, A. & Nordmann, P. (1999). Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum ß-lactamase VEB-1 in Pseudomonas aeruginosa. FEMS Microbiological Letters 176, 4119.[ISI][Medline]
15
.
Tribuddharat, C. & Fennewald, M. A. (1999). Integron-mediated rifampin resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 9602.
16 . Recchia, G. D. & Hall, R. M. (1995). Gene cassettes: a new class of mobile elements. Microbiology 141, 301527.[ISI][Medline]
17 . Hall, R. M. & Collis, C. M. (1995). Mobile gene cassettes and integrons: capture and spread of genes by site specific recombination. Molecular Microbiology 15, 593600.[ISI][Medline]
18 . Recchia, G. D. & Hall, R. M. (1998). Origins of the mobile gene cassettes found in integrons. Trends in Microbiology 5, 38994.[ISI]
19 . Collis, C. M. & Hall, R. M. (1997). Site-specific deletion and rearrangement of integron insert genes catalysed by the integron DNA integrase. Journal of Bacteriology 174, 157485.[Abstract]
20 . Stokes, H. W., O'Gorman, D. B., Recchia, G. D., Parsekhian, M. & Hall, R. M. (1997). Structure and function of 59-base element recombination sites associated with mobile gene cassettes. Molecular Microbiology 26, 73145.[ISI][Medline]
21 . Naas, T. & Nordmann, P. (1999). OXA-type ß-lactamases. Current Pharmaceutical Design 5, 86579.[ISI][Medline]
22
.
Poirel, L., Le Thomas, I., Naas, T., Karim, A. & Nordmann, P. (2000). Biochemical sequence analyses of GES-1, a novel class A extended-spectrum ß-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 44, 62232.
23 . Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 10319.[ISI][Medline]
24 . Danel, F., Hall, M. C., Gur, D. & Livermore, D. (1995). OXA-14, another extended-spectrum variant of OXA-10 (PSE-2) ß-lactamase from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 18814.[Abstract]
25 . Philippon, L. N., Naas, T., Bouthors, A. T., Barakett, V. & Nordmann, P. (1997). OXA-18, a class D clavulanic acid-inhibited extended-spectrum ß-lactamase from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 41, 218895.[Abstract]
26 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A3. NCCLS, Villanova, PA.
27 . Kieser, T. (1984). Factors affecting the isolation of cccDNA from Streptomyces lividans and Escherichia coli. Plasmid 12, 1936.[ISI][Medline]
28 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
29
.
Falbo, V., Caratolli, A., Tosini, F., Pezella, C., Dionisi, A. M. & Luzzi, I. (1999). Antibiotic resistance conferred by conjugative plasmid and a class 1 integron in Vibrio cholerae O1 El Tor strains isolated in Albania and Italy. Antimicrobial Agents and Chemotherapy 43, 6936.
30
.
Laraki, N., Galleni, M., Thamm, I., Riccio, M. L., Amicosante, G., Frère, J. M. et al. (1999). Structure of In31, a blaIMP-1-containing Pseudomonas aeruginosa integron phyletically related to In5, which carries an unusual array of gene cassettes. Antimicrobial Agents and Chemotherapy 43, 890901.
31 . Huovinen, P., Huovinen, S. & Jacoby, G. A. (1988). Sequence of PSE-2 ß-lactamase. Antimicrobial Agents and Chemotherapy 32, 1346.[ISI][Medline]
32 . Cameron, F. H., Groot Obbink, D. J., Ackennan, V. P. & Hall, R. M. (1986). Nucleotide sequence of the AAD(2') aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dfrII in R388. Nucleic Acids Research 14, 862535.[Abstract]
33 . Shaw, K. J., Rather, P. N., Hare, R. S. & Miller, G. H. (1993). Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Micobiological Reviews 57, 13863.
34
.
Ploy, M. C., Courvalin, P. & Lambert, T. (1998). Characterization of In40 of Enterobacter aerogenes BM2688, a class 1 integron with two novel gene cassettes, cmlA2 and qacF. Antimicrobial Agents and Chemotherapy 42, 255763.
35 . Paulsen, I. T., Littlejohn, T. G., Radström, P., Sundström, L., Sköld, O., Swedberg, G. et al. (1993). The 3' conserved segment of integrons contains a gene associated with multidrug resistance to antiseptics and disinfectants. Antimicrobial Agents and Chemotherapy 37, 7618.[Abstract]
36 . Paulsen, I. T., Skurray, R. A., Tam, R., Saier, M. H., Turner, R. J., Weiner, J. H. et al. (1996). The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs. Molecular Microbiology 19, 116775.[ISI][Medline]
37 . Kurokawa, H., Yagi, T., Shibata, N., Shibayama, K. & Arakawa, Y. (1999). Worldwide proliferation of carbapenem-resistant Gram-negative bacteria. Lancet 354, 955.[ISI][Medline]
38
.
Liu, P. Y., Tung, J. C., Ke, S. C. & Chen, S. L. (1998). Molecular epidemiology of extended spectrum ß-lactamase-producing Klebsiella pneumoniae isolates in a district hospital in Taiwan. Journal of Clinical Microbiology 36, 275962.
Received 27 January 2000; returned 26 April 2000; revised 19 June 2000; accepted 20 July 2000