1 Servicio Antimicrobianos, Dpto. Bacteriología, INEI-ANLIS Dr Carlos G. Malbrán, Av. Velez Sársfield 563 (1281), Buenos Aires; 2 Dpto. Microbiología, Facultad de Medicina, Universidad Nacional de Buenos Aires, Paraguay 2155, P. 12 (1120), Buenos Aires; 3 Servicio de Bacteriología, Hospital Gobernador Centeno, Calle 17 y 108, General Pico, Provincia de La Pampa, Argentina
Received 6 December 2002; returned 20 January 2003; revised 2 April 2003; accepted 7 April 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: CTX-M-2, PER-2, In35, Tn3, Tn1331
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
K. pneumoniae producing two or more ESBLs have been reported scarcely, but when they are, in most cases they include TEM- and/or SHV-derived enzymes.511 Herein we report the characterization of a multiresistant K. pneumoniae isolate that produces a novel combination of two ESBLs (CTX-M-2 and PER-2) and a derived variant of this strain, which was selected during ciprofloxacin treatment and showed alterations in the expression levels of outer membrane proteins (OMPs).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
K. pneumoniae M1803 and M1826 were isolated from a 1-year-old patient who suffered chronic urinary tract infection due to a posterior urethral valve malformation and bilateral vesicourethral reflux. During a period of 5 months, the child was treated for successive urinary infections with amphotericin (Gador, Buenos Aires, Argentina), ceftriaxone (Roche), furazolidone (nitrofurantoin analogue; Schering-Plough), amoxicillin (Roche), imipenem (imipenem/cilastatin; Merck Sharp & Dohme) and ciprofloxacin (Bayer). During one of these infections, K. pneumoniae strain M1803 was isolated. After 10 days of ciprofloxacin therapy, K. pneumoniae strain M1826 was isolated.
Escherichia coli ER1793 [F fhuA2 (lacZ)r1 glnV44 e14 (McrA) trp-31 his-1 rpsL104(StrR) xyl-7 mtl-2 metB1
(mcrC-mrr)114::IS10; New England Biolabs, Beverly, MA, USA], resistant to nalidixic acid and rifampicin, was employed as the recipient strain in conjugative assays.
Antimicrobial agents and susceptibility testing
The antimicrobial agents used for susceptibility testing were obtained from standard laboratory powders, and their sources were as follows: amikacin, aztreonam and cefepime (Bristol-Myers Squibb); amoxicillin and clavulanic acid (Smith Kline Beechman and Roemmers, Buenos Aires, Argentina); ampicillin (Temis Lostaló, Buenos Aires, Argentina); chloramphenicol (Parke Davis, Buenos Aires, Argentina); cefotaxime (Argentia, Buenos Aires, Argentina); cefoxitin and imipenem (without cilastatin) (Merck Sharp & Dohme); ceftazidime (Glaxo); ceftibuten, gentamicin, sulfamethoxazole and trimethoprim (Schering Plough); cefalothin, piperacillin and tazobactam (Wyeth Pharmaceuticals); tetracycline (Microsules and Bernabó, Buenos Aires, Argentina); meropenem (Astra Zeneca); and ciprofloxacin (Bagó, Buenos Aires, Argentina). The antimicrobial susceptibility of each isolate and its transconjugants was determined by the disc diffusion and agar dilution methods according to NCCLS guidelines.1215 When indicated, clavulanic acid (2 mg/L) or tazobactam (4 mg/L) was used in combination with ß-lactams.
Nalidixic acid and rifampicin (both for use in molecular biology applications) were from Sigma (St Louis, MO, USA).
Isoelectric focusing and microbiological method for detection of ß-lactamases
ß-Lactamase preparation and analytical isoelectric focusing (IEF) were performed as described previously.16 ß-Lactamase bands were visualized by the iodometric method described by Labia & Barthélémy.17 For ß-lactamase inhibition procedures the gel was covered with a piece of filter paper impregnated with 1 mM clavulanic acid, aztreonam or cefoxitin as indicated, and incubated for 10 min at room temperature before developing as described above. Crude preparations from bacteria possessing ß-lactamases of known isoelectric points (pI) were used as standards: TEM-1 (pI 5.4), PER-2 (pI 5.4), SHV-2 (pI 7.6), P99 (pI 7.8), CTX-M-2 (pI 7.9) and SHV-5 (pI 8.2).
The hydrolytic activity against cefoxitin and carbapenems was determined by the microbiological method.18
OMP analysis
OMPs were isolated as sodium lauroyl sarcosynate-insoluble material from cell envelopes after sonication (20 pulses of 30 s in ice at 30 s intervals) and centrifugation of lysed cells at 100 000g for 1 h at 4°C.19 Electrophoretic analysis of OMPs was performed as previously reported,19 using 12% SDSPAGE gels and broad molecular weight markers (unstained; Bio-Rad, Hercules, CA, USA) as standards.
Conjugative assays
Biparental conjugations were performed by mixing the donor and recipient strains on LuriaBertani agar in a ratio of 5:1, and incubating the mixture for 18 h at 35°C. Transconjugants were selected on MuellerHinton agar supplemented with nalidixic acid (50 mg/L) plus cefotaxime (8 mg/L), or nalidixic acid plus ceftazidime (15 mg/L).
General molecular biology procedures
The relatedness of K. pneumoniae M1803 and M1826 was assessed by pulsed-field gel electrophoresis (PFGE) of the XbaI-digested DNA using conditions already described.20 Plasmid DNA was extracted from K. pneumoniae and E. coli transconjugant cells as described previously,21 and were analysed by electrophoresis in 0.7% agarose gels (Trisacetate buffer), employing plasmids of known size as standards.
PCR assays
Detection of antimicrobial resistance genes was performed by PCR using the forward and reverse primers listed in Table 1. DNA templates were prepared by lysing one or two colonies of each K. pneumoniae isolate, or each E. coli transconjugant, in 50 µL of boiling water, and 2 µL was used for the PCRs. Amplifications were performed with a Biometra thermal cycler (Whatman Biometra GmbH, Göttingen, Germany) in a final volume of 50 µL containing 20 pmol of each primer (purchased from Gibco-BRL, Gaithersburg, MD, USA), 25 µM of each dNTP, 1.5 mM MgCl2 and 2.5 U of Taq polymerase (Promega, Madison, WI, USA). Previously described conditions for cycling were used.16,22,23
|
DNA sequencing of amplicons obtained with primers specific to blaCTX-M, blaPER and gyrA [including the region analogous to the quinolone resistance-determining region (QRDR)] genes (Table 1) was performed on both strands by the method of Sanger et al.,24 using the BigDye terminators methodology (Applied Biosystems/Perkin Elmer, Foster City, CA, USA). The sequences were analysed in an ABI Prism 377 DNA Sequencer (Applied Biosystems/Perkin Elmer).
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
K. pneumoniae M1803 and M1826 showed less than three differences according to PFGE profiling, indicating the clonal relationship between both isolates (Figure 1).25 In contrast to M1803, isolate M1826 was resistant to cefoxitin and ceftibuten, showing a 432x increase in the MICs of extended-spectrum cephalosporins, carbapenems, aztreonam, piperacillintazobactam, chloramphenicol and ciprofloxacin. In addition, clavulanic acid produced almost no reduction in the MICs of extended-spectrum cephalosporins against isolate M1826 (Table 2).
|
|
Characterization of ß-lactamases produced by K. pneumoniae M1803 and M1826
By IEF analysis, with the ß-lactamase bands being visualized by an iodometric method using ampicillin plus cefaloridine as substrate, three bands were detected in both K. pneumoniae strains, with pIs of 5.4, 7.6 (corresponding to the chromosomal SHV-1 ß-lactamase) and 7.9 (Table 2). In contrast, when visualizing the ß-lactamase bands with ceftazidime, two bands were detected, with pIs of 5.4 and 7.9. All bands were susceptible to clavulanic acid inhibition. The nature of a broad-spectrum ß-lactamase activity with a pI of 5.4 could not be specifically determined in these standard IEF assays because of an overlapping ESBL band at the same pI. Accordingly, in situ ß-lactamase inhibition using cefoxitin was performed on the IEF gel, as described previously.16 The ESBL activity with a pI of 5.4 was inhibited with cefoxitin, whereas the broad-spectrum ß-lactamase activity with a pI of 5.4 and the ESBL activity with a pI of 7.9 were not. This property, of cefoxitin inhibition of the ESBL band at pI 5.4, indicated it was most likely to be the PER-2 enzyme. These results are in agreement with those obtained by PCR assays, which yielded amplification products with primers specific to TEM, SHV, CTX-M and PER families of ß-lactamases genes. Therefore, the presence of four ß-lactamase genes, blaPER-2-like, blaCTX-M-2-like, blaSHV-1-like and blaTEM-1-like, was confirmed. These results were replicated using both types of transconjugant strains to determine which ß-lactamase gene is encoded for on which plasmid (Table 2). The identity of blaPER-2 and blaCTX-M-2 genes was unequivocally established by sequencing of the corresponding amplicons (data not shown).
Non-transferable antibiotic resistance caused by an OmpK35 and OmpK36 deficiency in K. pneumoniae M1826
K. pneumoniae isolates that are resistant to cefoxitin have been described previously, with resistance caused by either the production of a plasmid-mediated AmpC-like ß-lactamase26,27 or the loss of OMPs, such as OmpK35 and OmpK36.19,28 Indeed, reduced expression of both porins has been reported in K. pneumoniae strains with high MICs of cefoxitin (>32 mg/L).28 Moreover, increase in the levels of resistance to extended-spectrum cephalosporins, carbapenems and also fluoroquinolones other than cefoxitin, has been associated to the loss of both OmpK35 and OmpK36.19,29,30
In this study, the genetic determinants of K. pneumoniae M1826 conferring resistance to cefoxitin and carbapenems could not be transferred to E. coli by conjugation. Furthermore, incubation of the IEF gels with aztreonam (an AmpC-like ß-lactamase inhibitor), prior to ß-lactamase visualization using ampicillin plus cefaloridine, did not result in inhibition of any of the ß-lactamase bands produced by K. pneumoniae M1826 (data not shown). This fact suggests the absence of enzymic activity against cefoxitin in this isolate. Indeed, using a microbiological method, no enzymic activity against cefoxitin and carbapenems was detected in K. pneumoniae M1826. In this assay, the parallel control was a crude extract containing the known AmpC MIR-1, and this showed a positive result for cefoxitin hydrolysis. All this information suggests the absence of AmpC-type enzymes in K. pneumoniae M1826, and that cefoxitin resistance was chromosomal.
Accordingly, OMP analysis for K. pneumoniae M1803 and M1826 (cefoxitin MICs of 2 and 64 mg/L, respectively) was performed and showed that the latter was deficient in OmpK35/36 expression (Figure 2). Our results are in agreement with the previous reports mentioned above, suggesting that the deficiency in OmpK35/36 expression is responsible for the higher overall resistance levels displayed by the M1826 isolate (particularly to cefoxitin and carbapenems) as compared with those of the pretreatment M1803 isolate (Table 2).
|
Detailed analysis of the transferable resistance genes carried by K. pneumoniae M1803 and M1826
Very recently, the blaCTX-M-2 gene has been reported as being located in unusual class I integrons In35 and InS21, in clinical isolates of Proteus mirabilis and Salmonella enterica ser. Infantis, respectively.30,31 This unusual class 1 integron contains a partial duplication of the 3' conserved segment, and a region that includes blaCTX-M-2 and the Orf513 between both 3' conserved segments. In addition, In35-related integrons having blaCTX-M-2 in conjunction with different resistance cassettes such as aac(6')-Ib-ant(3'')-Ia, orfD, ant(2'')-Ia-ant(3'')-Ia, ant(3'')-Ia and aac(6')-Ib within the variable region of class 1 integrons were detected in clinical isolates of Enterobacteriaceae.30 In the present work, by PCR mapping of both K. pneumoniae isolates and the type 1 transconjugants, the blaCTX-M-2 gene was located in an unusual class 1 integron having ant(2'')-Ia-ant(3'')-Ia gene cassettes within the variable region (Figure 3a). The co-existence of blaCTX-M-2 and ant(2'')-Ia genes in the same genetic structure (a complex sulI-type integron) and harboured, in turn, in a conjugative megaplasmid, may explain the high prevalence in our bacterial population of both extended-spectrum cephalosporin and gentamicin resistance. The widespread distribution of CTX-M-2-like enzymes among clinical isolates of K. pneumoniae in Argentina (60%)4 could be explained by the fact that ceftriaxone and cefotaxime are the most used cephalosporins in our country. Another gene conferring resistance to gentamicin, aac(3)-IIa, was found in both type 1 and type 2 transconjugants. The finding of two gentamicin resistance genes [ant(2'')-Ia and aac(3)-IIa] in type 1 transconjugants correlated well with an MIC of gentamicin 8-fold higher than that observed for type 2 transconjugants (Table 2).
|
Conclusions
Generally, in Vibrio cholerae16 and Enterobacteriaceae4,33,34 from Argentina, ESBLs and TEM-1-like enzymes are encoded on the same megaplasmid, which, additionally, harbours genetic determinants conferring resistance to other kinds of antibiotics, such as aminoglycosides, trimethoprimsulfamethoxazole, chloramphenicol and tetracycline. Therefore, the use of any of these drugs in nosocomial acquired infections may lead to cross-selection of extended-spectrum cephalosporin resistance.
Worldwide, few descriptions of strains with two or more ESBLs have been reported, and all of those that have been reported involve TEM- or SHV-derived enzymes. We identified here two ESBLs, CTX-M-2 and PER-2, in a single multiresistant isolate, the first report of this ESBL combination in the same strain. The fact that blaCTX-M-2 and blaPER-2 were harboured in two different megaplasmids suggests a differential spread of these genes, which correlates well with their different prevalences (60% versus 10%, respectively4,33,34) observed in the Argentinean isolates. After successive antibiotic treatments, the last of them with ciprofloxacin, K. pneumoniae M1826 could be selected. The selection of these multiresistant bacteria in the hospital environment could be due, amongst other factors, to excessive and/or inappropriate antibiotic use, particularly of empirically prescribed broad-spectrum agents.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Du Bois, S., Marriott, M. & Amyes, S. (1995). TEM- and SHV-derived extended-spectrum ß-lactamases: relationship between selection, structure and function. Journal of Antimicrobial Chemotherapy 35, 722.[Abstract]
3 . Jacoby, G. A. & Medeiros, A. A. (1991). More extended-spectrum ß-lactamases. Antimicrobial Agents and Chemotherapy 35, 16971704.[ISI][Medline]
4 . Galas, M., Rapoport, M., Pasterán, F. et al. (1999). High distribution of CTX-M-2 ß-lactamase among Klebsiella spp. isolates in an Argentinean extended spectrum ß-lactamase (ESBLA) surveillance program. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 1474, p. 165. American Society for Microbiology, Washington, DC, USA.
5 . Bradford, P. A., Cherubin, C. E., Idemyor, V. et al. (1994). Multiply resistant Klebsiella pneumoniae strains from two Chicago hospitals: identification of the extended-spectrum TEM-12 and TEM-10 ceftazidime-hydrolyzing ß-lactamases in a single isolate. Antimicrobial Agents and Chemotherapy 38, 7616.[Abstract]
6
.
Essack, S., Hall, L., Pillay, D. et al. (2001). Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum ß-lactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa. Antimicrobial Agents and Chemotherapy 45, 8895.
7
.
Liu, P., Tung, J. C., Ke, S. C. et al. (1998). Molecular epidemiology of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae isolates in a district hospital in Taiwan. Journal of Clinical Microbiology 36, 275962.
8
.
Pai, H., Lyu, S., Lee, J. et al. (1999). Survey of extended-spectrum ß-lactamases in clinical isolates of Escherichia coli and Klebsiella pneumoniae: prevalence of TEM-52 in Korea. Journal of Clinical Microbiology 37, 175863.
9
.
Kim, J., Kwon, Y., Pai, H. et al. (1998). Survey of Klebsiella pneumoniae strains producing extended-spectrum ß-lactamases: prevalence of SHV-12 and SHV-2a in Korea. Journal of Clinical Microbiology 36, 14469.
10 . Soilleux, M., Morand, A., Arlet, G. et al. (1996). Survey of Klebsiella pneumoniae producing extended-spectrum ß-lactamases: prevalence of TEM-3 and first identification of TEM-26 in France. Antimicrobial Agents and Chemotherapy 40, 10279.[Abstract]
11
.
Yuan, M., Hall, L., Hoogkamp-Korstanje, J. et al. (2001). SHV-14, a novel ß-lactamase variant in Klebsiella pneumoniae isolates from Nijmegen, The Netherlands. Antimicrobial Agents and Chemotherapy 45, 30911.
12 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
13 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility TestingTwelfth Informational Supplement: MIC Testing, M100-S12. NCCLS, Wayne, PA, USA.
14 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Disk Susceptibility TestSeventh Edition: Approved Standard M2-A7. NCCLS, Wayne, PA, USA.
15 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility TestingTwelfth Informational Supplement: Disk Diffusion M100-S12. NCCLS, Wayne, PA, USA.
16
.
Petroni, A., Corso, A., Melano, R. et al. (2002). Plasmidic extended-spectrum ß-lactamases in Vibrio cholerae O1 El Tor in Argentina. Antimicrobial Agents and Chemotherapy 46, 14628.
17 . Labia, R. & Barthélémy, M. (1979). Lenzymogramme des beta-lactamases: adaptation en cel de la methode iodometrique. Annales de Microbiologie (Paris) 130B, 295304.
18 . Masuda, G., Tomioka, S. & Hasegawa, M. (1976). Detection of ß-lactamase production by gram-negative bacteria. Journal of Antibiotics (Tokyo) 29, 6624.[ISI][Medline]
19 . Martínez-Martínez, L., Hernández-Allés, S., Abertí, S. et al. (1996). In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum cephalosporins. Antimicrobial Agents and Chemotherapy 40, 3428.[Abstract]
20 . Foodborne and Diarrheal Diseases Branch, Centers for Disease Control and Prevention. (1998). Standarized Molecular Subtyping of Foodborne Bacterial Pathogens by Pulsed-Field Gel Electrophoresis: A Training Manual. Centers for Disease Control and Prevention, Atlanta, GA, USA.
21 . Nakamura, M., Sato, S., Ohya, T. et al. (1986). Plasmid profile analysis in epidemiological studies of animal Salmonella typhimurium infection in Japan. Journal of Clinical Microbiology 23, 3605.[ISI][Medline]
22 . Levesque, C., Piche, L., Larose, C. et al. (1995). PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrobial Agents and Chemotherapy 39, 18591.[Abstract]
23 . Arlet, G. & Philippon, A. (1989). Construction by polymerase chain reaction and intragenic DNA probes for three main types of transferable ß-lactamases (TEM, SHV, CARB). FEMS Microbiology Letters 82, 1926.
24 . Sanger, S., Micklen, S. & Coulson, A. (1977). DNA sequencing with chain-terminating inhibitors. Procedings of the National Academy of Sciences, USA 74, 54637.[Abstract]
25
.
Tenover, F. C., Arbeit, R. D., Goering, R. V. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 22339.
26 . González-Leiza, M., Pérez-Díaz, J., Ayala, J. et al. (1994). Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated ß-lactamase with two molecular variants. Antimicrobial Agents and Chemotherapy 38, 21507.[Abstract]
27 . Papanicolau, G. A., Medeiros, A. A. & Jacoby, G. A. (1990). Novel plasmid-mediated ß-lactamase (MIR-1) conferring resistance to oxyimino- and alpha-methoxy ß-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 34, 22009.[ISI][Medline]
28
.
Ardanuy, C., Liñares, J., Domínguez, M. et al. (1998). Outer membrane profiles of clonally related Klebsiella pneumoniae isolates from clinical samples and activities of cephalosporins and carbapenems. Antimicrobial Agents and Chemotherapy 42, 163640.
29
.
Martínez-Martínez, L., Pascual, A., Hernández-Allés, S. et al. (1999). Roles of ß-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 43, 166973.
30
.
Arduino, S. M., Roy, P. H., Jacoby, G. A. et al. (2002). blaCTX-M-2 is located in an unusual class 1 integron (In35) which includes Orf513. Antimicrobial Agents and Chemotherapy 46, 23036.
31
.
Di Conza, J., Ayala, J. A., Power, P. et al. (2002). Novel class 1 integron (InS21) carrying blaCTX-M-2 in Salmonella enterica serovar Infantis. Antimicrobial Agents and Chemotherapy 46, 225761.
32 . Tolmasky, M. & Crosa, J. (1993). Genetic organization of antibiotic resistance genes (aac(6')-Ib, aadA, and oxa9) in the multiresistance transposon Tn1331. Plasmid 29, 3140.[CrossRef][ISI][Medline]
33 . Galas, M. F., Pasterán, F. G., Melano, R. G. et al. (1998). Unusual distribution of enzymatic resistance to third-generation cephalosporin (TGC) in E. coli in Argentina. In Program and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract E-109, p. 201. American Society for Microbiology, Washington, DC, USA.
34 . Pasterán, F. G., Melano, R. G., Galas, M. F. et al. (1999). High proportion of extended spectrum ß-lactamases (ESBLA) among AmpC producers enterobacteria in Argentina. In Program and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 1475, p. 166. American Society for Microbiology, Washington, DC, USA.