Journal of Antimicrobial Chemotherapy (1999) 44, 377-380
© 1999 The British Society for Antimicrobial Chemotherapy
Brief report |
Molecular characterization of a multiply resistant Klebsiella pneumoniae encoding ESBLs and a plasmid-mediated AmpC
Center for Research in Anti-Infectives and Biotechnology,Departments of Pediatrics and Medical Microbiology and Immunology, Creighton University School of Medicine and Methodist Hospital, 2500 California Plaza, Omaha, NE 68178, USA
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Abstract |
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Organisms encoding multiple antibiotic resistance genes are becoming increasingly prevalent. In this report we describe a multiply resistant Klebsiella pneumoniae which possesses at least five different ß-lactamase genes. Isoelectric focusing, polymerase chain reaction and restriction fragment length polymorphism analysis identified TEM-1, multiple SHVs, OXA-9 and a plasmid-mediated ampC ß-lactamase. Furthermore, Southern analysis and conjugation experiments established that most of the resistance genes were encoded on one large transferable plasmid. This report demonstrates the complexity of multiply resistant organisms.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The emergence and spread of plasmid-mediated ß-lactamases such as extended-spectrum ß-lactamases (ESBLs), AmpC ß-lactamases and inhibitor-resistant derivatives of TEM and SHV have been potentiated by the use and over-use of broad-spectrum ß-lactam antibiotics.1 The genes encoding these ß-lactamases are often located on large plasmids that also encode genes for resistance to other antibiotics, including aminoglycosides, tetracycline, sulfonamides, trimethoprim and chloramphenicol.1 Furthermore, there is an increasing tendency for pathogens to produce multiple ß-lactamases.1 In this report, we describe an isolate of Klebsiella pneumoniae which is multiply resistant and expresses a minimum of five different ß-lactamases, most of which are encoded on one large transferable plasmid.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Patient history
K. pneumoniae 225 was isolated from a severe laceration to the head of a 43-year-old white male working in a New York City sewage canal on 26 April, 1996. Initially the wound, which extended 20 cm back from the left forehead and down to the skull, was surgically debrided, irrigated and closed. The patient was given preoperative cefazolin and gentamicin intravenously, with cefazolin continued 24 h post-operatively. Several weeks later, the patient experienced significant swelling and pain in the left scalp area, significant weakness and dizziness, and low grade fever. The wound was surgically drained and empirical treatment with piperacillin/tazobactam and ciprofloxacin was begun. Therapy was changed to imipenem/cilastatin after receipt of antibiotic susceptibility results indicating susceptibility only to this agent and fluoroquinolones, and resistance to all other tested ß-lactams, aminoglycosides, trimethoprim/sulphamethoxazole, tetracycline and chloramphenicol. After 4 weeks of therapy, all signs of inflammation resolved and the patient remained free from infection at follow-up on 10 December, 1996.
Susceptibility testing and isoelectric focusing
Susceptibility tests were performed on K. pneumoniae 225 by NCCLS microdilution methodology in Mueller–Hinton broth (CM 405, Oxoid, Basingstoke, UK) using an inoculum of approximately 5 x 105 cfu/mL2 and by NCCLS disc diffusion.2
Isoelectric focusing (IEF), cefotaxime hydrolysis and inhibitor determinations in polyacrylamide gels were performed using sonic extracts of K. pneumoniae 225.3
Polymerase chain reaction (PCR)
Template DNA preparation and PCR amplifications were carried out as described previously, except that Triton X-100 was deleted from the PCR reaction mixture, the total volume was 50 µL, and 2 µL of template DNA and an annealing temperature of 55°C were used.4 The following oligonucleotide primer sets specific for the TEM or SHV gene families, Enterobacter ampC and OXA-9 were used in a PCR: TEM, (forward) 5'-AGATCAGTTGGGTGCACGAG-3' [nucleotides (nt) 313–332], (reverse) 5'-TGCTTAATCAGTGAGGCACC-3' (nt 1061–1042); SHV, (forward) 5'-GGGAAACGGAACTGAATGAG-3' (nt 606–625), (reverse) 5'-ATCGTCCACCATCCACTGCA-3' (nt 757–738); OXA-9, (forward) 5'-CGTCGCTCACCATATCTCCC-3' (nt 2783–2802), (reverse) 5'-CCTCTCGTGCTTTAGACCCG-3' (nt 3097–3078); and Enterobacter ampC, (forward) 5'-ATTCGTATGCTGGATCTCGCCACC-3' (nt 413–436), (reverse) 5'-CATGACCCAGTTCGCCATATCCTG-3' (nt 808–785). 5 ,6 ,7 SHV-specific PCR products were sequenced by automated PCR cycle-sequencing with dye-terminator chemistry using a DNA stretch sequencer from Applied Biosystems (Foster City, CA, USA).
Plasmid DNA and Southern analysis
Plasmid DNA was isolated using alkaline lysis8 with the following modifications: cell pellets were washed twice with 3% Triton X-100 dissolved in Tris–EDTA, pH 8. After neutralization, supernatant was extracted with phenol plus 1/10 volume 10% SDS followed by one extraction using phenol:chloroform:isoamyl alcohol (25:24:1) followed by one or more chloroform:isoamyl alcohol (24:1) extractions until the supernatant was clear. Chromosomal DNA was digested with plasmid-safe DNase (Epicentre Technologies, Madison, WI, USA). Plasmids were separated by agarose (0.8%) gel electrophoresis using 1 x TAE as the buffer system.
Southern analysis was performed on plasmid DNA isolated by alkaline lysis and separated as described above. DNA was transferred using 0.4 M NaOH to Zeta-Probe blotting membranes. TEM- (5'-TGCTTAATCAGTGAGGCACC-3'; nt 1062–10426) and SHV- (5'-TTAGCGTTGCCAGTGCTCG-3'; nt 988–9706) specific probes were labelled using the Genius System Oligonucleotide 3'-end labelling kit (Boehringer Mannheim, Indianapolis, IN, USA). Prehybridization and hybridization followed the recommendation of the manufacturer using 1% SDS at 37°C. Blots were washed using tetramethylammonium chloride (TMAC) once at 37°C for 15 min and twice at 48°C for 20 min. Labelled probe hybridized to plasmid DNA was detected using the Genius Luminescent detection kit (Boehringer Mannheim).
DNA was transformed using a modified Hanahan method (TSS) described by Clontech (Clontech Laboratories, Inc. Transformer site. Directed Mutagenesis Kit—2nd version, Palo Alto, CA, USA) into Escherichia coli HB101. Conjugation experiments were carried out by filter mating using E. coli strain C600N (Nalr) as the recipient. Transconjugants were selected on Luria–Bertani agar plates containing 30 mg/L of nalidixic acid. An indole test was performed on the transconjugant (E. coli C600N) to differentiate it further from the donor (K. pneumoniae 225).
Restriction fragment length polymorphism (RFLP)
SHV-specific PCR products were used directly in an NheI restriction endonuclease assay.9 Fragments were resolved using 2% agarose and a 1x TAE buffer system.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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In this study, an isolate of K. pneumoniae was identified which contained a minimum of five different ß-lactamase genes and possessed other drug resistance mechanisms as well. The MICs were as follows:
64 mg/L of ticar-cillin, co-amoxiclav,
ticarcillin/clavulanate, piperacillin, piperacillin/tazobactam, ceftazidime, cefixime, loracarbef,
cephalothin, cefazolin, cefoxitin, aztreonam, ampicillin/sulbactam and cefpodoxime; 16 mg/L of
cetriaxone and cefotaxime; 1 mg/L of imipenem and cefepime; 0.5 mg/L of ciprofloxacin; 0.06
mg/L of meropenem; >256 mg/L of sulphamethoxazole; >64 mg/L of nitrofurantoin;
>32 mg/L of amikacin; >8 mg/L of gentamicin, tobramycin and tetracycline; and >2/38
mg/L of trimethoprim/sulphamethoxazole, respectively. The MIC values of the ß-lactam
drugs
and the results of IEF analysis suggested the expression of ESBLs and a plasmid-mediated
AmpC. Five ß-lactamase bands with pI values of 5.4, 6.8, 7.6, 8.2 and 9.0 were
identified (Table). The ß-lactamase bands corresponding to pI values
of 7.6, 8.2 and 9.0
hydrolysed cefotaxime. In addition, the band corresponding to pI value 9.0 was resistant to
clavulanate. These results suggested the presence of clavulanate-sensitive ESBLs of pI values 7.6
and 8.2, and an AmpC enzyme with a pI value 9.0.
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PCR analysis was used initially to confirm and/or identify the ß-lactamases observed during isoelectric focusing (Table). PCR identified the presence of TEM and SHV genes, an Enterobacter ampC gene and the OXA-9 gene (Table). SHV-specific PCR products were sequenced and identified the presence of SHV-5.
Profiles of plasmid DNA isolated from K. pneumoniae 225, treated with plasmid-safe DNase, showed that the organism carried two plasmids of 17 kb and approximately 90 kb (data not shown). Southern analysis revealed that both encoded TEM-derived genes. However, only the 90 kb plasmid encoded the SHV-derived genes (data not shown).
In an attempt to isolate each plasmid, transformation experiments were carried out. As it was possible to transform the 17 kb plasmid but not the 90 kb plasmid, conjugation experiments were also performed. Conjugation between K. pneumoniae 225 and E. coli C600N resulted in the transfer of both the 90 and 17 kb plasmids. Disc susceptibilities of the parent K. pneumoniae 225 strain, the E. coli transconjugant containing the 90 kb plasmid and the E. coli transformant were compared. The resistance pattern for the transconjugant was similar to the resistance pattern of the parent K. pneumoniae. Both of these strains were resistant to ceftriaxone, ceftazidime, cefotetan, cefoxitin, piperacillin/tazobactam, sulphamethoxazole with trimethoprim, aztreonam, gentamicin and chloramphenicol. The transformant was resistant to only ampicillin and amikacin. Therefore, cefoxitin resistance of the transconjugant, but not the transformant strongly suggested that the gene encoding ampC was located on the 90 kb plasmid. These data together with the Southern analysis suggest that most of the resistance mechanisms encoded by K. pneumoniae were encoded on the 90 kb plasmid.
Some ESBL-SHV enzymes with a pI of 7.6 or 8.2 contain a glycine to serine amino acid substitution at position 238 due to a mutation which creates a new endonuclease restriction site, NheI.6,9 For example, this restriction site is not present in the structural genes of ESBLs SHV-6 or SHV-8, or the structural genes of broad-spectrum ß-lactamases (BSBLs), SHV-1 or SHV-11.9 The initial development of the NheI RFLP analysis of SHV genes was capable of distinguishing ESBL-SHVs from BSBL-SHVs.9 However, the published sequence of SHV-6 and the identification of SHV-11 and SHV-12 no longer makes this differentiation valid.6 In organisms that encode multiple ß-lactamase genes, the combination of IEF and RFLP analysis of SHV-specific PCR products can aid in the identification of multiple SHV-type ß-lactamases encoded by one isolate. To help determine whether all SHV genes encoded by K. pneumoniae 225 carried the glycine to serine mutation, RFLP analysis on SHV-specific PCR products from K. pneumoniae 225 was performed using NheI. The presence of the NheI site in the SHV-specific PCR product will result in two bands of 219 and 164 bp. The absence of the NheI site will result in no cleavage and a full-length fragment of 383 bp. In the figure, lanes 1 and 2 represent SHV-specific PCR products from a clinical isolate expressing SHV-1. As expected, in the presence of NheI (lane 2), no cleavage was observed. However, lanes 3 and 4 and 7 and 8 represent PCR products amplified from isolates which express SHV-4 and SHV-7, respectively. In each case, the enzyme NheI was able to cleave these bands resulting in two bands of 219 and 164 bp, indicative of the mutation. SHV-specific PCR products amplified from template prepared from K. pneumoniae 225 showed both full-length and cleaved products (Figure, lane 6). Therefore, some of the SHV-encoding gene products contain the amino acid substitution at position 238 of glycine to serine and some do not. The full-length PCR product which was refractory to restriction by NheI probably represents SHV-1. It has been suggested that SHV-1 is encoded by chromosomal DNA in K. pneumoniae; therefore, its presence would be expected.10 The cleaved products could represent SHV-2 and/or SHV-5. Sequence analysis of SHV-specific PCR products confirmed the presence of SHV-5; therefore, the IEF band of pI 8.2 represents SHV-5, indicating that the IEF band of pI 7.6 capable of cefotaxime hydrolysis probably represented two ß-lactamase enzymes, SHV-1 and SHV-2.
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It is intriguing to speculate about the epidemiology of the K. pneumoniae isolate and its multidrug resistance encoding plasmid. The early onset of signs of infection upon his return to Omaha suggests that the patient acquired the organism in New York, where the administration of drugs to which it was resistant (cefazolin and gentamicin) would have provided a selective pressure that facilitated its multiplication in the sewage-contaminated lesion. The occurrence of multidrug resistance encoding plasmids in isolates of K. pneumoniae from medical environments in which there is heavy antibiotic usage has been extensively documented, supporting a hypothesis that the original source of the isolate may have been a medical setting.1,4,10However, little is known about the selective pressures and the extent of gene transfer within sewage microflora. Therefore, the possibility of the sewer system as the source of this multiply resistant organism should not be overlooked.
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Acknowledgments |
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We thank Stacey Morrow for excellent technical assistance. This research was supported in part by funds from the Center for Research in Anti-Infectives and Biotechnology. DNA sequencing was supported in part by UNMC/Eppley Cancer Center grant P30CA36727.
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Notes |
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Corresponding author. Tel: +1-402-280-1881; Fax: +1-402-280-1225; E-mail: ndhanson{at}creighton.edu
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Received 20 November 1998; returned 10 February 1999; revised 20 March 1999; accepted 27 April 1999
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