Prevalence of Derepressed AmpC Mutants and Extended-Spectrum -Lactamase Producers among Clinical Isolates of Citrobacter freundii, Enterobac
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微生物临床杂志 2005年第5期
Department of Microbiology, Dankook University College of Medicine, Cheonan, Republic of Korea
ABSTRACT
The resistance mechanism of extended-spectrum cephalosporins in clinical isolates of Citrobacter freundii, Enterobacter spp., and Serratia marcescens was studied. Of 152 isolates, 45 isolates (29.6%) were derepressed AmpC mutants and 39 isolates (25.7%) produced extended-spectrum -lactamase (ESBLs). The most prevalent ESBLs were CTX-M enzymes, followed by TEM-52 and SHV-12.
TEXT
In recent years, extended-spectrum -lactamases (ESBLs) have become more and more prevalent in species characterized by inducible class C cephalosporinase (AmpC), such as Enterobacter spp., Citrobacter freundii, or Serratia marcescens, which frequently segregate mutants with high-level constitutive production of AmpC enzymes (5, 8-9, 12, 16). Although less common than AmpC hyperproduction, ESBLs among these species are a problem of great concern due to the potential transmission of resistance to other bacterial species and because ESBLs are usually encoded by plasmids that also harbor genes for resistance to non--lactam antibiotics such as aminoglycosides (13, 17).
In Korea, 47% and 51% of Enterobacter cloacae and S. marcescens isolates, respectively, were resistant to cefotaxime and resistance rates to ceftazidime were 48% and 25%, respectively, in the 1998 survey (4). Although the resistance rates of E. cloacae and S. marcescens to third-generation cephalosporins are considerably high, studies on the resistance mechanism of extended-spectrum cephalosporins among these species have been rarely performed in Korea. Therefore, we aimed to study the resistance mechanism of extended-spectrum cephalosporins in clinical isolates of C. freundii, Enterobacter spp., and S. marcescens and determine the prevalence rate of derepressed AmpC mutants or ESBL-producing organisms among these species.
Between June and November 2003 from three university hospitals located in three different cities in Korea, 152 consecutive nonduplicate nosocomial isolates, including 21 isolates of C. freundii, 15 of E. aerogenes, 44 of E. cloacae, and 72 of S. marcescens, were collected. All isolates were subjected to the double-disk synergy test (6), isoelectric focusing (10), antimicrobial susceptibility test (11), and PCR and sequencing for -lactamase genes with the primers in Table 1.
According to the characteristics of -lactamase production, we defined the isolates as follows: derepressed AmpC mutants are those with a cefoxitin MIC of 32 mg/liter and a cefotaxime MIC of 32 mg/liter and without ESBL production; ESBL producers are isolates which produced ESBLs regardless of the cefoxitin or cefotaxime MIC; and inducible AmpC-producing strains are those with a cefotaxime MIC of 16 mg/liter and without ESBL production. When we categorized the 152 clinical isolates of C. freundii, Enterobacter spp., and S. marcescens according to the definition described above, 45 (29.6%) isolates were derepressed AmpC mutants, 39 (25.7%) isolates were ESBL producers, and 68 (44.7%) isolates were inducible AmpC-producing strains (Table 2).
Distribution of ESBLs among the clinical isolates of C. freundii, Enterobacter spp., and S. marcescens are shown in Table 3. The most prevalent ESBL types were CTX-M type ESBLs, which have been rarely found in Korea, followed by TEM-52 and SHV-12. Two C. freundii isolates produced a novel SHV-type -lactamase with a pI of 7.6, and the bla gene encoding this enzyme differs from that encoding blaSHV-1 by only one amino acid change, at position 54 (Asp Glu). Because these two isolates were positive by the double-disk synergy test, we regarded this enzyme as an ESBL.
ESBLs among the isolates of Enterobacter spp., C. freundii, and S. marcescens have been described from several countries worldwide and become more and more prevalent (1, 2, 5, 9, 12-14, 16, 17). Initially, the ESBLs among these species were typical TEM or SHV enzymes (9, 13, 16), but enzymes of the CTX-M class have been described more recently (1-3, 5, 8, 12). The CTX-M-3 enzyme was the most common ESBL type among the S. marcescens isolates in Poland (12), and CTX-M-9 and CTX-M-14 were detected in E. cloacae isolates from China (3). In Korea, TEM-52, SHV-2a, and SHV-12 are the most prevalent ESBL types among the family Enterobacteriaceae and CTX-M type ESBLs have been rarely found (7, 14-15). But in this study, CTX-M enzymes such as CTX-M-3, CTX-M-9, and CTX-M-15 were commonly identified among the isolates of Enterobacter spp. and S. marcescens, suggesting dissemination of the enzymes among these species. To our knowledge, this is the first report of CTX-M-3, CTX-M-9, and CTX-M-15 among the clinical isolates of Enterobacter spp., C. freundii, and S. marcescens in Korea and of CTX-M-15 in E. aerogenes isolates in the world.
The MIC ranges and MICs at which 50% of the isolates tested are inhibited (MIC50s) of several -lactam antibiotics are given in Table 4. For cefepime, 33% (13 of 39) of isolates producing ESBLs were nonsusceptible to cefepime whereas only 6% (3 of 45) of derepressed AmpC mutants were nonsusceptible to cefepime. The data suggest that cefepime still has activity against derepressed mutants but not against ESBL producers. Thus, in order to use cefepime safely for the treatment of infections by Enterobacter spp., Citrobacter, or S. marcescens, differentiation of ESBL producers from derepressed mutants is necessary for those clinical isolates.
In 45 strains of derepressed mutants, the resistance rate to cefotetan was 98%, but none of 68 isolates of inducible strains were resistant to cefotetan. Pai et al. (14) also showed similar data that the MIC of cefotetan was 32 mg/liter for 94% of derepressed or partially derepressed AmpC mutants but the MIC of cefotetan was 16 mg/liter for 96.5% of inducible strains regardless of ESBL production. Therefore, susceptibility to cefotetan would help to differentiate derepressed mutants from inducible strains.
Antimicrobial susceptibilities to trimethoprim, ciprofloxacin, chloramphenicol, tetracycline, and aminoglycosides such as tobramycin, gentamicin, and amikacin were also examined. ESBL production was associated with high levels of resistance to trimethoprim and ciprofloxacin compared to both derepressed chromosomal AmpC mutants and inducible AmpC mutants. The resistance rates of ESBL producers to trimethoprim, ciprofloxacin, tobramycin, and gentamicin were very high at 54%, 72%, 95%, and 74%, respectively. Since ESBL producers express their -lactamase genes from plasmids, these findings suggest that genes coding for ESBLs and genes coding for resistance to these antibiotics may reside within the same plasmids and therefore be spread together. This means that resistance to two different kinds of drugs may be coselected by the use of either one and all of these antimicrobials could be a selective pressure for spreading of such isolates.
ACKNOWLEDGMENTS
We are grateful to the following people, who supplied the clinical isolates used in this study: Je-Chul Lee and Sung-Yong Seol, Kyungpook National University School of Medicine, and Insoo Rheem, Dankook University College of Medicine.
This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (03-PJ1-PG1-CH03-0002).
REFERENCES
Baraniak, A., J. Fiett, A. Sulikowska, W. Hryniewicz, and M. Gniadkowski. 2002. Countrywide spread of CTX-M-3 extended-spectrum beta-lactamase-producing microorganisms of the family Enterobacteriaceae in Poland. Antimicrob. Agents Chemother. 46:151-159.
Canton, R., A. Oliver, T. M. Coque, M. C. Varela, J. C. Perez-Diaz, and F. Baquero. 2002. Epidemiology of extended-spectrum -lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period. J. Clin. Microbiol. 40:1237-1243.
Chanawong, A., F. H. M'Zail, J. Heritage, J. H. Xiong, and P. M. Hawkey. 2002. Three cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's Republic of China. Antimicrob. Agents Chemother. 46:630-637.
Chong, Y., and K. Lee. 2000. Present situation of antimicrobial resistance in Korea. J. Infect. Chemother. 6:189-195.
De Champs, C., D. Sirot, C. Chanal, R. Bonnet, J. Sirot, and The French Study Group. 2000. A 1998 survey of extended-spectrum beta-lactamases in Enterobacteriaceae in France. Antimicrob. Agents Chemother. 44:3177-3179.
Jarlier, V., M. Nicolas, G. Fournier, and A. Philippon. 1988. Extended broad-spectrum -lactamases conferring transferable resistance to newer -lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev. Infect. Dis. 10:867-878.
Kim, J., Y. Kwon, H. Pai, J. W. Kim, and D. T. Cho. 1998. Survey of Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases: prevalence of SHV-12 and SHV-12a in Korea. J. Clin. Microbiol. 36:1446-1449.
Livermore, D. M. 1995. -Lactamases in laboratory and clinical resistance. Clin. Microbiol. Rev. 4:557-584.
Livermore, D. M., and D. F. J. Brown. 2001. Detection of -lactamase-mediated resistance. J. Antimicrob. Chemother. 48:S59-S64.
Mathew, A., A. M. Harris, M. J. Marshall, and G. W. Ross. 1975. The use of analytical isoelectric focusing for detection and identification of -lactamases. J. Gen. Microbiol. 88:169-178.
National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard, 5th ed., p. 7-10. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Naumiuk, L., A. Baraniak, M. Gniadkowski, B. Krawczyk, B. Rybak, E. Sadowy, A. Samet, and J. Kur. 2004. Molecular epidemiology of Serratia marcescens in two hospitals in Danzig, Poland, over a 5-year period. J. Clin. Microbiol. 42:3108-3116.
Neuwirth, C., E. Siebor, J. Lopez, A. Pechinot, and A. Kazmierczak. 1996. Outbreak of TEM-24-producing Enterobacter aerogenes in an intensive care unit and dissemination of the extended-spectrum -lactamase to other members of the family Enterobacteriaceae. J. Clin. Microbiol. 34:76-79.
Pai, H., J. Y. Hong, J. H. Byeon, Y. K. Kim, and H. J. Lee. 2004. High prevalence of extended-spectrum beta-lactamase-producing strains among blood isolates of Enterobacter spp. collected in a tertiary hospital during an 8-year period and their antimicrobial susceptibility patterns. Antimicrob. Agents Chemother. 48:3159-3161.
Pai, H., E. H. Choi, H. J. Lee, J. Y. Hong, and G. A. Jacoby. 2001. Identification of CTX-M-14 extended-spectrum -lactamase in clinical isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea. J. Clin. Microbiol. 39:3747-3749.
Paterson, D. L. 2001. Extended-spectrum beta-lactamases: the European experience. Curr. Opin. Infect. Dis. 14:697-701.
Pitout, J. D., K. S. Thomson, N. D. Hanson, A. F. Ehrhardt, P. Coudron, and C. C. Sanders. 1998. Plasmid-mediated resistance to expanded-spectrum cephalosporins among Enterobacter strains. Antimicrob. Agents Chemother. 42:596-600.(Jungmin Kim and Yu-Mi Lim)
ABSTRACT
The resistance mechanism of extended-spectrum cephalosporins in clinical isolates of Citrobacter freundii, Enterobacter spp., and Serratia marcescens was studied. Of 152 isolates, 45 isolates (29.6%) were derepressed AmpC mutants and 39 isolates (25.7%) produced extended-spectrum -lactamase (ESBLs). The most prevalent ESBLs were CTX-M enzymes, followed by TEM-52 and SHV-12.
TEXT
In recent years, extended-spectrum -lactamases (ESBLs) have become more and more prevalent in species characterized by inducible class C cephalosporinase (AmpC), such as Enterobacter spp., Citrobacter freundii, or Serratia marcescens, which frequently segregate mutants with high-level constitutive production of AmpC enzymes (5, 8-9, 12, 16). Although less common than AmpC hyperproduction, ESBLs among these species are a problem of great concern due to the potential transmission of resistance to other bacterial species and because ESBLs are usually encoded by plasmids that also harbor genes for resistance to non--lactam antibiotics such as aminoglycosides (13, 17).
In Korea, 47% and 51% of Enterobacter cloacae and S. marcescens isolates, respectively, were resistant to cefotaxime and resistance rates to ceftazidime were 48% and 25%, respectively, in the 1998 survey (4). Although the resistance rates of E. cloacae and S. marcescens to third-generation cephalosporins are considerably high, studies on the resistance mechanism of extended-spectrum cephalosporins among these species have been rarely performed in Korea. Therefore, we aimed to study the resistance mechanism of extended-spectrum cephalosporins in clinical isolates of C. freundii, Enterobacter spp., and S. marcescens and determine the prevalence rate of derepressed AmpC mutants or ESBL-producing organisms among these species.
Between June and November 2003 from three university hospitals located in three different cities in Korea, 152 consecutive nonduplicate nosocomial isolates, including 21 isolates of C. freundii, 15 of E. aerogenes, 44 of E. cloacae, and 72 of S. marcescens, were collected. All isolates were subjected to the double-disk synergy test (6), isoelectric focusing (10), antimicrobial susceptibility test (11), and PCR and sequencing for -lactamase genes with the primers in Table 1.
According to the characteristics of -lactamase production, we defined the isolates as follows: derepressed AmpC mutants are those with a cefoxitin MIC of 32 mg/liter and a cefotaxime MIC of 32 mg/liter and without ESBL production; ESBL producers are isolates which produced ESBLs regardless of the cefoxitin or cefotaxime MIC; and inducible AmpC-producing strains are those with a cefotaxime MIC of 16 mg/liter and without ESBL production. When we categorized the 152 clinical isolates of C. freundii, Enterobacter spp., and S. marcescens according to the definition described above, 45 (29.6%) isolates were derepressed AmpC mutants, 39 (25.7%) isolates were ESBL producers, and 68 (44.7%) isolates were inducible AmpC-producing strains (Table 2).
Distribution of ESBLs among the clinical isolates of C. freundii, Enterobacter spp., and S. marcescens are shown in Table 3. The most prevalent ESBL types were CTX-M type ESBLs, which have been rarely found in Korea, followed by TEM-52 and SHV-12. Two C. freundii isolates produced a novel SHV-type -lactamase with a pI of 7.6, and the bla gene encoding this enzyme differs from that encoding blaSHV-1 by only one amino acid change, at position 54 (Asp Glu). Because these two isolates were positive by the double-disk synergy test, we regarded this enzyme as an ESBL.
ESBLs among the isolates of Enterobacter spp., C. freundii, and S. marcescens have been described from several countries worldwide and become more and more prevalent (1, 2, 5, 9, 12-14, 16, 17). Initially, the ESBLs among these species were typical TEM or SHV enzymes (9, 13, 16), but enzymes of the CTX-M class have been described more recently (1-3, 5, 8, 12). The CTX-M-3 enzyme was the most common ESBL type among the S. marcescens isolates in Poland (12), and CTX-M-9 and CTX-M-14 were detected in E. cloacae isolates from China (3). In Korea, TEM-52, SHV-2a, and SHV-12 are the most prevalent ESBL types among the family Enterobacteriaceae and CTX-M type ESBLs have been rarely found (7, 14-15). But in this study, CTX-M enzymes such as CTX-M-3, CTX-M-9, and CTX-M-15 were commonly identified among the isolates of Enterobacter spp. and S. marcescens, suggesting dissemination of the enzymes among these species. To our knowledge, this is the first report of CTX-M-3, CTX-M-9, and CTX-M-15 among the clinical isolates of Enterobacter spp., C. freundii, and S. marcescens in Korea and of CTX-M-15 in E. aerogenes isolates in the world.
The MIC ranges and MICs at which 50% of the isolates tested are inhibited (MIC50s) of several -lactam antibiotics are given in Table 4. For cefepime, 33% (13 of 39) of isolates producing ESBLs were nonsusceptible to cefepime whereas only 6% (3 of 45) of derepressed AmpC mutants were nonsusceptible to cefepime. The data suggest that cefepime still has activity against derepressed mutants but not against ESBL producers. Thus, in order to use cefepime safely for the treatment of infections by Enterobacter spp., Citrobacter, or S. marcescens, differentiation of ESBL producers from derepressed mutants is necessary for those clinical isolates.
In 45 strains of derepressed mutants, the resistance rate to cefotetan was 98%, but none of 68 isolates of inducible strains were resistant to cefotetan. Pai et al. (14) also showed similar data that the MIC of cefotetan was 32 mg/liter for 94% of derepressed or partially derepressed AmpC mutants but the MIC of cefotetan was 16 mg/liter for 96.5% of inducible strains regardless of ESBL production. Therefore, susceptibility to cefotetan would help to differentiate derepressed mutants from inducible strains.
Antimicrobial susceptibilities to trimethoprim, ciprofloxacin, chloramphenicol, tetracycline, and aminoglycosides such as tobramycin, gentamicin, and amikacin were also examined. ESBL production was associated with high levels of resistance to trimethoprim and ciprofloxacin compared to both derepressed chromosomal AmpC mutants and inducible AmpC mutants. The resistance rates of ESBL producers to trimethoprim, ciprofloxacin, tobramycin, and gentamicin were very high at 54%, 72%, 95%, and 74%, respectively. Since ESBL producers express their -lactamase genes from plasmids, these findings suggest that genes coding for ESBLs and genes coding for resistance to these antibiotics may reside within the same plasmids and therefore be spread together. This means that resistance to two different kinds of drugs may be coselected by the use of either one and all of these antimicrobials could be a selective pressure for spreading of such isolates.
ACKNOWLEDGMENTS
We are grateful to the following people, who supplied the clinical isolates used in this study: Je-Chul Lee and Sung-Yong Seol, Kyungpook National University School of Medicine, and Insoo Rheem, Dankook University College of Medicine.
This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (03-PJ1-PG1-CH03-0002).
REFERENCES
Baraniak, A., J. Fiett, A. Sulikowska, W. Hryniewicz, and M. Gniadkowski. 2002. Countrywide spread of CTX-M-3 extended-spectrum beta-lactamase-producing microorganisms of the family Enterobacteriaceae in Poland. Antimicrob. Agents Chemother. 46:151-159.
Canton, R., A. Oliver, T. M. Coque, M. C. Varela, J. C. Perez-Diaz, and F. Baquero. 2002. Epidemiology of extended-spectrum -lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period. J. Clin. Microbiol. 40:1237-1243.
Chanawong, A., F. H. M'Zail, J. Heritage, J. H. Xiong, and P. M. Hawkey. 2002. Three cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's Republic of China. Antimicrob. Agents Chemother. 46:630-637.
Chong, Y., and K. Lee. 2000. Present situation of antimicrobial resistance in Korea. J. Infect. Chemother. 6:189-195.
De Champs, C., D. Sirot, C. Chanal, R. Bonnet, J. Sirot, and The French Study Group. 2000. A 1998 survey of extended-spectrum beta-lactamases in Enterobacteriaceae in France. Antimicrob. Agents Chemother. 44:3177-3179.
Jarlier, V., M. Nicolas, G. Fournier, and A. Philippon. 1988. Extended broad-spectrum -lactamases conferring transferable resistance to newer -lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev. Infect. Dis. 10:867-878.
Kim, J., Y. Kwon, H. Pai, J. W. Kim, and D. T. Cho. 1998. Survey of Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases: prevalence of SHV-12 and SHV-12a in Korea. J. Clin. Microbiol. 36:1446-1449.
Livermore, D. M. 1995. -Lactamases in laboratory and clinical resistance. Clin. Microbiol. Rev. 4:557-584.
Livermore, D. M., and D. F. J. Brown. 2001. Detection of -lactamase-mediated resistance. J. Antimicrob. Chemother. 48:S59-S64.
Mathew, A., A. M. Harris, M. J. Marshall, and G. W. Ross. 1975. The use of analytical isoelectric focusing for detection and identification of -lactamases. J. Gen. Microbiol. 88:169-178.
National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard, 5th ed., p. 7-10. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Naumiuk, L., A. Baraniak, M. Gniadkowski, B. Krawczyk, B. Rybak, E. Sadowy, A. Samet, and J. Kur. 2004. Molecular epidemiology of Serratia marcescens in two hospitals in Danzig, Poland, over a 5-year period. J. Clin. Microbiol. 42:3108-3116.
Neuwirth, C., E. Siebor, J. Lopez, A. Pechinot, and A. Kazmierczak. 1996. Outbreak of TEM-24-producing Enterobacter aerogenes in an intensive care unit and dissemination of the extended-spectrum -lactamase to other members of the family Enterobacteriaceae. J. Clin. Microbiol. 34:76-79.
Pai, H., J. Y. Hong, J. H. Byeon, Y. K. Kim, and H. J. Lee. 2004. High prevalence of extended-spectrum beta-lactamase-producing strains among blood isolates of Enterobacter spp. collected in a tertiary hospital during an 8-year period and their antimicrobial susceptibility patterns. Antimicrob. Agents Chemother. 48:3159-3161.
Pai, H., E. H. Choi, H. J. Lee, J. Y. Hong, and G. A. Jacoby. 2001. Identification of CTX-M-14 extended-spectrum -lactamase in clinical isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea. J. Clin. Microbiol. 39:3747-3749.
Paterson, D. L. 2001. Extended-spectrum beta-lactamases: the European experience. Curr. Opin. Infect. Dis. 14:697-701.
Pitout, J. D., K. S. Thomson, N. D. Hanson, A. F. Ehrhardt, P. Coudron, and C. C. Sanders. 1998. Plasmid-mediated resistance to expanded-spectrum cephalosporins among Enterobacter strains. Antimicrob. Agents Chemother. 42:596-600.(Jungmin Kim and Yu-Mi Lim)