JAC Advance Access originally published online on September 22, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):1048-1053; doi:10.1093/jac/dkl370
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New antibiotic resistance genes associated with CTX-M plasmids from uropathogenic Nigerian Klebsiella pneumoniae
1 Department of Pharmaceutical Microbiology, University of Ibadan Ibadan, Nigeria 2 Department of Pathobiology, School of Public Health and Community Medicine, University of Washington Seattle, WA 98195, USA
*Corresponding author. Tel: +1-206-543-8001; Fax: +1-206-543-3873; E-mail: marilynr{at}u.washington.edu
Received 26 June 2006; returned 25 July 2006; revised 8 August 2006; accepted 15 August 2006
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Abstract |
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Objectives: To determine antibiotic resistance genes associated with 17 Nigerian CTX-M-positive Klebsiella pneumoniae plasmids from patients with community-acquired urinary tract infections.
Methods: The size and restriction patterns of the plasmids were determined, and antibiotic resistance genes were identified using DNA–DNA hybridization, PCR assays, hybridization of PCR products with internal probes, and sequencing.
Results: All CTX-M plasmids were large (58–320 kb) and carried the following genes: aac(6')-Ib (aminoglycoside resistance) which included aac(6')-Ib-cr (aminoglycoside-fluoroquinolone resistance), aadA2 (aminoglycoside resistance), erm(B) (macrolide-lincosamide-streptogramin B resistance), blaTEM-1 (ampicillin resistance), tet(A) (tetracycline resistance), sul1 (sulphonamide resistance), dfr (trimethoprim resistance) and intI1, an integrase associated with class 1 integrons. Eleven (65%) plasmids carried an mph(A) gene (macrolide resistance), seven (41%) plasmids carried a qnrB1 gene (low-level quinolone resistance) and four (24%) plasmids carried multiple cat genes (chloramphenicol resistance). catA2, catA3 and qnrB1 genes and a 6 kb PstI fragment, carrying the blaCTX-M gene, were sequenced.
Conclusions: This is the first description of catA2 and catA3 genes in Klebsiella spp. and the first description of the erm(B) and floR genes associated with a CTX-M plasmid. This is also the first report of qnrB1 and aac(6')-Ib-cr in isolates from Africa and the first report of these two genes on the same plasmid.
Keywords: ESBLs , plasmids , multidrug resistance genes
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The CTX-M ß-lactamases are one of the groups of extended-spectrum ß-lactamases (ESBLs) identified after the introduction of the broad-spectrum cephalosporins. These enzymes have spread worldwide and 54 different types have been identified in the past 10 years (http://www.lahey.org.studies, 23 June 2006, date last accessed). We recently reported on 30 ß-lactamase-positive Nigerian Klebsiella pneumoniae of which 17 carried CTX-M ß-lactamases (blaCTX-M-1group gene).1 The blaCTX-M genes were located on large plasmids (58–320 kb) that were transformed into Escherichia coli and resulted in multiresistant transformants.1 In the present study, we identified other antibiotic resistance genes carried on these CTX-M plasmids, including the first description of catA2 and catA3 genes in K. pneumoniae, the first description of the rRNA methylase gene erm(B) and floR genes on a CTX-M plasmid, the first detection of the qnrB1 and the aac(6')-Ib-cr genes in isolates from Africa and the first report of the qnrB1 and the aac(6')-Ib-cr genes on the same plasmid.
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Materials and methods |
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Bacterial isolates
Seventeen E. coli DH5
transformants carrying one CTX-M-positive plasmid (58–320 kb) described previously were used.1 The sizes of the plasmids were estimated by electrophoresis on 0.7% agarose using the plasmids from E. coli V517 as the standard markers2,3 and from PstI and BamHI restriction patterns.
Antibiotic susceptibility
Susceptibilities to nalidixic acid, florfenicol and trimethoprim/sulfamethoxazole were determined by disc diffusion on Mueller–Hinton agar (Remel Inc, Lenexa, KS, USA) according to Clinical and Laboratory Standards Institute (CLSI) guidelines with the E. coli ATCC 25922 control.4 MICs of erythromycin were determined using the agar dilution method as described previously.5
Conjugation
Conjugation experiments were carried out using E. coli HB101 as the recipient as described previously.5 Transconjugants were selected on Luria–Bertani agar (Difco Laboratories, Kansas, MO, USA) supplemented with rifampicin (25 mg/L) and tetracycline (25 mg/L), or chloramphenicol (25 mg/L) as described previously.5
Plasmid DNA restriction analysis
Plasmid DNA was extracted with the Qiagen midi-preps kit (Qiagen Inc., Germantown, MD, USA), or a modified alkaline lysis procedure, digested with either PstI or BamHI and electrophoresed through 0.7% agarose gel as described previously.6 The reference plasmids from E. coli V517 were obtained using an in-well mild cell lysis to obtain the 166.7 and 344 kb plasmids3 while the modified alkaline lysis procedure was used to obtain eight other plasmids from E. coli V517 (2.2–58 kb).2
Detection of antibiotic and mercury resistance genes
DNA–DNA hybridization was used to screen for the resistance genes, while PCR assays with hybridization of the PCR products using internal 32P-labelled probes and sequencing of selected amplicons was used for verification of the following antibiotic resistance genes: chloramphenicol resistance genes catA1, catA2, catA3 and catB3; florfenicol resistance gene floR; macrolide resistance genes erm(B), ere(A), ere(B), mph(A), mph(B) and mph(D); plasmid-mediated quinolone resistance genes qnrA, qnrB and qnrS; orf513 and orf1005 previously associated with the qnr-type genes;7,8 the intI1 integrase; sul1 coding for sulphonamide resistance; trimethoprim resistance dfr genes; aminoglycoside resistance genes aadA2, aac(6')-Ib, armA and rmtA; and the merA gene coding for resistance to inorganic mercury as described previously.5 The PCR primers and probes are listed in Table 1.
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Cloning and sequencing
The CTX-M plasmid from Kpn19 was digested with PstI enzyme using the manufacturer’s conditions (New England BioLabs, Ipswich, MA, USA). The CTX-M-positive 6 kb fragment identified was cloned into the PstI site of pACYC177 and transformed into E. coli DH5 with selection of cefotaxime resistance (plasmid pMRC152).1 The 6 kb fragment was completely sequenced (AY995206
[GenBank]
). The complete sequence was also determined for catA2, catA3 and qnrB1 genes (DQ489998
[GenBank]
, DQ489999
[GenBank]
and DQ777878
[GenBank]
, respectively). Two selected aac(6')-Ib PCR products, from a qnrB1-positive and a qnrB1-negative plasmid, were sequenced to determine whether the plasmids contained the aac(6')-Ib-cr mutations (DQ303918
[GenBank]
). All sequences were determined as described previously.1
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
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The 17 CTX-M plasmids were restricted with PstI, which resulted in three distinct patterns. The first group included 12 plasmids with 4 bands, the second group included 4 plasmids with 7 bands and the third group included 1 plasmid with 11 bands (data not shown). The 12 plasmids with indistinguishable PstI patterns could be further subdivided into five different BamHI patterns which were labelled type A–E. Eight of the plasmids had indistinguishable BamHI and PstI patterns, though they did carry different antibiotic resistance genes. Two plasmids had indistinguishable BamHI patterns from each other, but differed from the first eight plasmids, one conferred chloramphenicol resistance and the other did not and were labelled type B. The remaining seven plasmids each had distinct BamHI patterns and were labelled types C-I. Six transformants were all able to transfer the CTX-M plasmid to a susceptible recipient creating a multidrug-resistant transconjugant in the laboratory (Table 2).
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Each of the 17 plasmids had a common 6 kb PstI fragment that contained the blaCTX-M-1 gene (data not shown). A cloned 6 kb fragment (pMRC152) was sequenced (AY458016 [GenBank] ) and found to be indistinguishable from a previously published 6 kb region identified in plasmid pC15-Ia (AY995206 [GenBank] ) from a CTX-M-15 E. coli outbreak in Canada.9 The ISEcp1 element, located in the 6 kb fragment, belongs to the IS1380 family (http://www-is.biotoul.fr/is.html) and has recently been shown to mobilize the adjacent blaCTX-M-19 gene.10 Finding the ISEcp1 element upstream of the blaCTX-M-15 gene in the Nigerian plasmid is consistent with the hypothesis that the ISEcp1 element is responsible for the wide distribution of the blaCTX-M gene.10
The 17 CTX-M-positive K. pneumoniae were all chloramphenicol resistant and carried multiple cat genes including catA2 and catA3 (data not shown), but only four of the E. coli transformants were chloramphenicol resistant (Table 2). All four chloramphenicol-resistant plasmids had distinct BamHI restriction patterns (B, C, E and H) and carried four cat genes, catB3, catA1 catA2 and catA3 (Table 2). One catA2 gene and one catA3 gene from transformant #7 were sequenced (DQ489998 [GenBank] and DQ489999 [GenBank] ) and found to be indistinguishable at the amino acid level from previously sequenced catA2 (DQ267820 [GenBank] ) and catA3 (AJ319822 [GenBank] ). The floR gene was also detected in the four chloramphenicol-resistant transformants by PCR and hybridization of PCR products with 32P-labelled internal probes (Table 2). This is the first description of the carriage of multiple cat genes in K. pneumoniae. A previous study described the carriage of multiple cat genes in a chloramphenicol-resistant Salmonella isolated in the People’s Republic of China.11
The 17 transformants had erythromycin MICs of 256–>512 mg/L and all 17 plasmids carried the erm(B) gene; 11 (65%) plasmids carried the mph(A) gene, while none of the plasmids carried the other five macrolide resistance genes tested (Table 2). This is the first time that either the erm(B) or the mph(A) genes have been associated with a CTX-M plasmid. The mph(A) gene had previously been associated with blaTEM-1 of TnSF1, a Tn21-like transposon in Shigella flexneri (AF188331 [GenBank] ).
Recently the qnr genes, which confer low-level quinolone resistance, have been identified on CTX-M plasmids from strains isolated in the United States and South India7,8 with the first qnrB1-positive plasmid isolated from an Indian K. pneumoniae CTX-M-15-positive plasmid.8 Seven (41%) of the Klebsiella plasmids carried qnrB1. The gene from the plasmid isolated from transformant #1 was sequenced (DQ777878 [GenBank] ) and found to be indistinguishable from previously sequenced qnrB1 (DQ351241 [GenBank] ). In contrast, the 17 plasmids were negative for the qnrA and qnrS genes (data not shown). Eleven plasmids, including the seven qnrB1-positive isolates, carried the orf1005 gene, previously found near the qnrB1 gene,8 while all plasmids were negative for the orf513 gene previously associated with other qnr genes.7,8
A PCR assay previously shown to amplify any variant of aac(6')-Ib12 produced an amplicon of the right size for all 17 transformants and we sequenced amplicons from two plasmids (one qnrB1-positive transformant #1 and one qnrB1-negative transformant #10). Both carried the two mutations (Trp-102
Arg and Asp-179Tyr) that are associated with fluoroquinolone and aminoglycoside resistance (Table 2).12 All 17 plasmids carried the sul1 and intI1 integrase genes, which are both associated with the class 1 integrons13 (Table 2). None of the plasmids carried aminoglycoside resistance genes armA or rmtA.14,15 Fifteen (88%) plasmids were positive by the dfrA1/A15/A16 PCR assay and two sequenced amplicons (transformants #28 and #29) were indistinguishable from the dfrA16 gene, and two sequenced amplicons were indistinguishable from the dfrA17 gene (transformant #15) (Table 2). The merA gene, coding for resistance to inorganic mercury, was identified in seven (41.2%) of the plasmids and conferred to their host the ability to grow on fresh medium supplemented with 27.152 mg/L HgCl2 (Sigma-Aldrich Co., St Louis, MO, USA) while the remaining E. coli transformants did not grow on mercury-supplemented medium (Table 2).
The present study reports the first description of the qnrB1 and aac(6')-Ib-cr genes from African isolates and is the first report of the qnrB1 and aac(6')-Ib-cr genes on the same plasmid. This is the first report of an association between the CTX-M plasmids and erm(B), floR, mph(A), catA2 and catA3 genes and the first report of catA2 and catA3 genes in Klebsiella spp. as well as the first description of multiple cat genes in K. pneumoniae.
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TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
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None to declare.
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Acknowledgements |
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In memory of Professor Herbert A. Odelola, Department of Pharmaceutical Microbiology, University of Ibadan, Ibadan, Nigeria.
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References |
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1 Soge OO, Queenan AM, Ojo KK, et al. (2006) CTX-M-15 extended-spectrum ß-lactamase from Nigerian Klebsiella pneumoniae. J Antimicrob Chemother 57:24–30.
2 Macrina FL, Kopecko DJ, Jones KR, et al. (1978) A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1:417–20.[CrossRef][Web of Science][Medline]
3 Pedraza RO and Diaz Ricci JC. (2002) In-well cell lysis technique reveals two new mega plasmids of 103.0 and 212.6 MDa in the multiple plasmid-containing strain V517 of Escherichia coli. Lett Appl Microbiol 34:130–3.[CrossRef][Web of Science][Medline]
4 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests—Eighth Edition: Approved Standard M2-A8 (2003) (NCCLS, Wayne, PA, USA).
5
Ojo KK, Ulep C, Van Kirk N, et al. (2004) The mef(A) gene predominates among seven macrolide resistant genes identified in 13 Gram-negative genera from healthy Portuguese children. Antimicrob Agents Chemother 48:3451–6.
6 Alcantar-Curiel D, Tinoco JC, Gayosco C, et al. (2004) Nosocomial bacteremia and urinary tract infections caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae with plasmids carrying both SHV-5 and TLA-1 genes. Clin Infect Dis 38:1067–74.[CrossRef][Web of Science][Medline]
7
Wang M, Sahm DF, Jacoby GA, et al. (2004) Emerging plasmid-mediated quinolone resistance associated with the qnr gene in Klebsiella pneumoniae clinical isolates in the United States. Antimicrob Agents Chemother 48:1295–9.
8
Jacoby GA, Walsh KE, Mills DM, et al. (2006) qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Agents Chemother 50:1178–82.
9
Boyd DA, Tyler S, Christianson S, et al. (2004) Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum ß-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob Agents Chemother 48:3758–64.
10
Poirel L, Decousser JW, Nordmann P. (2003) Insertion sequence ISEcp1B is involved in expression and mobilization of a blaCTX-M ß-lactamase gene. Antimicrob Agents Chemother 47:2938–4.
11
Chen S, Zhao S, White DG, et al. (2004) Characterization of multiple-antimicrobial-resistant Salmonella serovars isolated from retail meats. Appl Environ Microbiol 70:1–7.
12 Robicsek A, Strahileyitz J, Jacoby GA, et al. (2006) Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 12:83–8.[CrossRef][Web of Science][Medline]
13
Huovinen P, Sundstrom L, Swedberg G, et al. (1995) Trimethoprim and sulfonamide resistance. Antimicrob Agents Chemother 39:279–89.
14
Galimand M, Courvalin P, Lambert T. (2003) Plasmid-mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation. Antimicrob Agents Chemother 47:2565–71.
15 Yokoyama K, Doi Y, Yamane K, et al. (2003) Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Lancet 362:1888–93.[CrossRef][Web of Science][Medline]
16
Frech G, Kehrenberg C, Schwarz S. (2003) Resistance phenotypes and genotypes of multiresistant Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen isolates from animal sources. J Antimicrob Chemother 51:180–2.
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