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Heterogeneity of blaIND metallo-β-lactamase-producing Chryseobacterium indologenes isolates detected in Hefei, China
International Journal of Antimicrobial Agents 32 (2008) 398–400
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Heterogeneity of blaIND metallo--lactamase-producing Chryseobacterium indologenes isolates detected in Hefei, China Xiang-hong Lin a , Yuan-hong Xu a,∗ , Jun Cheng b , Tao Li a , Zhong-xin Wang a a b
Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China Department of Infectious Diseases, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
a r t i c l e
i n f o
Article history: Received 19 January 2008 Accepted 27 April 2008 Keywords: Chryseobacterium indologenes Carbapenem Resistance Genotype Metallo--lactamase
a b s t r a c t This study investigated the phenotypic and genetic properties of 28 clinical isolates of metallo-lactamase (MBL)-producing Chryseobacterium indologenes recovered from a university hospital. Twenty isolates were confirmed to carry a blaIND gene. Among them, 9 isolates were confirmed to carry the IND-1 gene, 10 contained the IND-2 gene and 1 had blaIND-3 alleles. One strain (No. 7) confirmed to be carrying the blaIND-1 gene was susceptible to imipenem and was negative in phenotypic methods. The data revealed that the antimicrobial resistance patterns of C. indologenes harbouring MBL genes are remarkably distinct. Two specific types of MBL genes, blaIND-1 and blaIND-2 , were identified to be the major genotype for C. indologenes isolated from Hefei, China. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Over the last few years, interest in Chryseobacterium spp. has grown rapidly due to the emergence of multidrug-resistant Chryseobacterium spp. Chryseobacterium spp. are intrinsically resistant to many -lactams, most commonly associated with the production of high levels of metallo--lactamases (MBLs). Chryseobacterium indologenes is a species commonly reported to cause various clinical syndromes [1]. Production of biofilm on foreign materials as well as protease activity may play an important role in the virulence of invasive infections due to C. indologenes [2]. Chryseobacterium indologenes isolates usually possess MBLs [3], which confer resistance to carbapenems such as imipenem (IPM) and meropenem (MEM). A specific type of MBL, BlaIND, has been identified in isolates of C. indologenes, with more than ten blaIND alleles identified and registered in GenBank. MBL-producing Gram-negative organisms, including Chryseobacterium meningosepticum, have recently been described in China [4,5]. Little is known about the distribution and heterogeneity of blaIND in clinical isolates of C. indologenes. The aim of the present study was to assess the prevalence and types of MBL-positive bacteria among 28 clinical C. indologenes isolated between March 2004 and December 2006 in Hefei, China. Moreover, the phenotypes and
∗ Corresponding author. Tel.: +86 551 292 3175; fax: +86 551 365 8417. E-mail address: [email protected] (Y.-h. Xu).
genetic properties of MBL-producing C. indologenes strains were also determined. 2. Materials and methods 2.1. Bacterial strains and antimicrobial susceptibility testing Twenty-eight clinically isolated C. indologenes were collected between March 2004 and December 2006 at the First Affiliated Hospital of Anhui Medical University, a 1500-bed public tertiary care teaching hospital in Hefei, China. All isolates were nonduplicates and were re-identified using MicroScan WA40 (Dade Behring Corp., Deerfield, IL). Minimum inhibitory concentration determination of antimicrobial agents was performed by the agar dilution method according to the Clinical and Laboratory Standards Institute [6]. Selected drugs active against Gram-positive bacteria were tested against C. indologenes and the results were interpreted according to breakpoints approved for staphylococci [7]. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as quality control strains. 2.2. Phenotypic detection of MBLs Two phenotypic detection methods, the three-dimensional (3D) test and the IPM–ethylene diamine tetraacetic acid (EDTA) (2mercaptopropionic acid) disc synergy test, were designed to screen for MBLs. For the 3D tests, crude enzyme extracts were prepared
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.04.028
X.-h. Lin et al. / International Journal of Antimicrobial Agents 32 (2008) 398–400
from isolates by cell sonication. Crude enzyme extract only (30 L), enzyme extract (30 L) plus 2 L of 2-mercaptopropionic acid and enzyme extract (30 L) plus 5 L of 0.5 M EDTA (pH 8.0) were each loaded in slots of a Mueller–Hinton agar plate. One IPM disc (10 g) (Oxoid, Basingstoke, UK) was placed in the centre of the plate. Escherichia coli ATCC 25922 was inoculated as the indicator isolate. Additionally, screening for MBLs was performed by the disc approximation test using a modification of the procedure described by Arakawa et al. [8] and Yong et al. [9]. Three IPM discs were placed 30 mm apart (centre to centre) on a Mueller–Hinton agar plate, onto which C. indologenes was spread. One IPM disc contained 5 L of 0.5 M EDTA (pH 8.0) and one contained 2 L of 2-mercaptopropionic acid. Pseudomonas aeruginosa 119/05 (VIM-2 producer) was used as a positive control. 2.3. Polymerase chain reaction (PCR) amplification and DNA sequencing PCR analysis was performed on all isolates to confirm the presence of MBL genes. blaIMP-1 , blaVIM-1 , blaVIM-2 , blaSPM-1 and blaIND genes were assayed using previously reported primers [3,10]. Four pairs of primers that amplify the whole blaIND gene were also used (Table 1). Nucleotide automated sequencing of both strands of the PCR products derived with primers that amplify the whole blaIND gene was performed with an ABI Prism 3730 DNA sequencer (Perkin-Elmer, Applied Biosystems Division, Foster City, CA). 2.4. Conjugation, plasmid preparation, ˇ-lactamase assays and isoelectric focusing Conjugation experiments were carried out with streptomycinresistant E. coli C600 strains as recipients. Transconjugants were selected on Mueller–Hinton agar plates containing 100 g/mL streptomycin and 2 g/mL IPM. Plasmid DNA of C. indologenes isolates producing MBL was extracted using QIAquick Gel Extraction Kit (QIAGEN GmbH, Hilden, Germany) and was used as the template DNA in PCR for the detection of blaIND . Carbapenemase activity in cell sonicates from overnight Luria–Bertani broth cultures was determined using a spectrophotometer (UV-SP752; Spectrum Corp., Shanghai, China) as described previously [11]. Inhibition by EDTA was assayed as described previously [11]. The protein content of the sonicates was determined using a commercial assay reagent (Bio-Rad, Foster City, CA) with bovine serum albumin as a standard. Determination of the isoelectric point (pI) of -lactamases was performed in pre-cast 5% polyacrylamide gels containing ampholytes (pH range 3.5–9.5; Ampholine PAGplate; Amersham Biosciences, Uppsala, Sweden) using nitrocefin chromogenic substrate for zymographic detection of -lactamases, as described Table 1 Polymerase chain reaction (PCR) primers used in analysis of the blaIND gene Primer
Sequence (5 –3 )
Amplicon (bp)
Annealing (◦ C)
blaIND Univ
F-GCCCAGGTTAAAGATTTTGTAAT R-CATGGCCACCGCCTTTCCATTC F-ATGAAAAAAAGCATCCG R-CTATTTTTTATTCAGAAGTTC F-ATGAAAAAAAGTATTCAGCTTTTG R-TTATTCCGGCTTTTTATTCTTATC F-ATGAAAAAAAGAATTCAGTTC R-TTATTTTTTGTTAAGAAGTTC F-ATGAGGAAAAATGTTAGGA R-CTACTGTTTTTGTTGATCTAA
580
52
720
48
732
49
720
48
720
49
blaIND1 group blaIND2 group blaIND3 group blaIND4 group
399
previously [11]. TEM-1 (pI 5.4), SHV-1 (pI 7.6), SHV-5 (pI 8.2) and AmpC (pI 8.8) were used as controls.
3. Results and discussion In the antimicrobial susceptibility test, most of the 28 C. indologenes strains were resistant to piperacillin, IPM, MEM, cefotaxime, aztreonam and ceftazidime, showing resistance rates >50% (Table 2). Significantly, 23 (82%) were resistant to IPM and MEM, which are widely used to treat infections caused by multidrugresistant Gram-negative bacteria. Isolates in this study show greater susceptibility to levofloxacin (71%), ciprofloxacin (68%) and gatifloxacin (75%). Moreover, vancomycin and rifampicin, which are more frequently used for Gram-positive bacteria, are an even better choice. Twenty C. indologenes isolates were confirmed to carry a blaIND gene, including 9 isolates carrying blaIND-1 , 10 carrying blaIND-2 and 1 carrying blaIND-3 alleles (Table 2). Among them, eight strains were isolated from respiratory tract specimens, four were isolated from blood, three were from urine and the remaining five were isolated from various other specimens. None of the isolates had other MBL genes except for blaIND . The majority of blaIND -producers isolated from this area had either blaIND-1 or blaIND-2 . IND-2 shared 80% amino acid identity with IND-1 and had a very similar broadspectrum resistance profile, and they were classified in functional subgroup 3a of class B carbapenem-hydrolysing -lactamases [3]. In the current study, two isolates, Nos. 10 and 16, harboured blaIND-1 and blaIND-2 , respectively. Their gene sequences contained mutations and were identified to be two new blaIND alleles, which were registered and deposited in GenBank under accession numbers EF394436 and EF394439. Interestingly, of the 20 blaIND -carrying isolates, 19 were found to be positive in two phenotypic MBL detection methods. One C. indologenes isolate (No. 7) gave a negative result both with the 3D test and the disc synergy test. Overall, the sensitivity and specificity of the results in different methods were 100% and 95%, respectively. Nineteen (83%) of the 23 IPM-resistant isolates harboured a blaIND gene. However, repeated attempts to transfer the MBL gene by conjugation failed. Plasmids were extracted from three C. indologenes strains producing MBLs but none of them harboured MBL genes. The results indicate that blaIND genes are very likely located on the chromosome. With the exception of isolate No. 7, cell sonicates of all blaIND positive isolates hydrolysed IPM in spectrophotometric assays and this activity was inhibited by EDTA. Using extracts of four C. indologenes strains randomly, the mean IPM-hydrolysing activity was ca. 70 mol/min/mg of protein. Meanwhile, isoelectric focusing of 19 blaIND -positive isolate extracts (not including No. 7) detected a -lactamase with a pI of 8.6–8.8, in keeping with IND-type lactamase. Other pI values could not be detected. The naturally occurring resistance to many antimicrobial agents such as aztreonam may not be linked to other -lactamases in C. indologenes. Although four isolates were highly resistant to IPM in the current study, we failed to amplify blaIND , which may indicate that other carbapenem resistance mechanisms are present in C. indologenes isolates. On the other hand, isolate No. 7 was confirmed to be carrying blaIND-1 but was susceptible to IPM and MEM. We found no phenotypic evidence of MBL production for this strain. This phenomenon of low-level resistance to carbapenems for other MBL-producing Gram-negative bacteria was confirmed, as previously described [12]. The variation in the permeability coefficient for each -lactam may play a major role. Further investigations for C. indologenes No. 7 are necessary to evaluate the role of permeability coefficient changes.
400
X.-h. Lin et al. / International Journal of Antimicrobial Agents 32 (2008) 398–400
Table 2 Minimum inhibitory concentrations (MICs) of antibiotics for Chryseobacterium indologenes isolates with various metallo--lactamase (MBL) genes No.
Origin
MBL
1 3 4 6 7 9 10 12 14 16 17 18 19 20 22 24 25 26 27 28
Urine Blood Sputum Sputum Blood Pus Sputum Sputum Stool Wound Sputum Wound Sputum Urine Blood Sputum Sputum Blood Urine Pus
blaIND-1 blaIND-2 blaIND-2 blaIND-2 blaIND-1 blaIND-2 blaIND-1 blaIND-1 blaIND-1 blaIND-2 blaIND-2 blaIND-1 blaIND-2 blaIND-1 blaIND-2 blaIND-2 blaIND-3 blaIND-1 blaIND-2 blaIND-1
MIC (mg/L) PIP
CAZ
CIP
GAT
ATM
IPM
MEM
LVX
CTX
TZP
RIF
VAN
128 >128 64 >128 32 >128 >128 64 128 >128 >128 >128 64 >128 128 >128 >128 128 64 >128
64 >128 32 >128 8 >128 128 128 64 32 >128 16 4 >128 >128 128 >128 32 16 64
0.25 4 0.25 2 0.25 8 16 2 4 0.5 >32 0.5 0.25 2 4 16 8 >32 0.25 0.5
1 2 0.5 4 1 32 8 0.5 2 1 8 0.5 1 8 16 4 8 16 0.5 1
>128 128 32 >128 16 >128 >128 128 128 64 >128 128 32 128 >128 128 >128 64 64 128
32 128 64 128 4 64 >128 32 64 64 >128 64 32 >128 128 64 128 128 32 64
64 >128 64 128 4 32 128 32 32 128 >128 32 32 >128 128 32 64 >128 64 64
2 16 1 4 0.5
>128 128 32 4 8 32 16 4 32 128 >128 64 64 >128 128 64 128 >128 16 32
8 32 4 16 8 32 64 4 8 32 >128 2 4 64 8 8 16 4 4 8
0.5 2 1 32 4 4 16 2 4 16 64 2 2 4 8 8 4 64 1 2
4 4 2 64 16 64 128 8 8 32 128 4 8 32 64 64 16 32 2 4
8 1 2 1 32 0.5 1 1 2 8 2 4 1 0.5
PIP, piperacillin; CAZ, ceftazidime; CIP, ciprofloxacin; GAT, gatifloxacin; ATM, aztreonam; IPM, imipenem; MEM, meropenem; LVX, levofloxacin; CTX, cefotaxime; TZP, piperacillin–tazobactam; RIF, rifampicin; VAN, vancomycin.
BlaIND, like BlaB and GOB from C. meningosepticum, confirmed the variability of chromosome-located class B -lactamases [3,13]. Chryseobacterium meningosepticum expresses two non-related MBLs, BlaB and GOB, with genetic and biochemical heterogeneity. In contrast to C. meningosepticum, C. indologenes may possess only one type of MBL, BlaIND. Although the prevalence of C. indologenes is low, it seems clear that the proportion of MBL-producing and multidrug-resistant isolates in our collection was higher than has been reported for C. meningosepticum in other parts of the world [4,7]. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Hsueh PR, Teng LJ, Yang PC, Ho SW, Hsieh WC, Luh KT. Increased incidence of nosocomial Chryseobacterium indologenes infections in Taiwan. Eur J Clin Microbiol Infect Dis 1997;16:568–74. [2] Pan HJ, Teng LJ, Chen YC, Hsueh PR, Yang PC, Ho SW, et al. High protease activity of Chryseobacterium indologenes isolates associated with invasive infection. J Microbiol Immunol Infect 2000;33:223–6. [3] Bellais S, Poirel L, Leotard S, Naas T, Nordmann P. Genetic diversity of carbapenem-hydrolyzing metallo--lactamases from Chryseobacterium (Flavobacterium) indologenes. Antimicrob Agents Chemother 2000;44:3028–34. [4] Chen GX, Zhang R, Zhou HW. Heterogeneity of metallo-beta-lactamases in clinical isolates of Chryseobacterium meningosepticum from Hangzhou, China. J Antimicrob Chemother 2006;57:750–2.
[5] Yu YS, Qu TT, Zhou JY, Wang J, Li HY, Walsh TR. Integrons containing the VIM-2 metallo--lactamase gene among imipenem-resistant Pseudomonas aeruginosa strains from different Chinese hospitals. J Clin Microbiol 2006;44: 4242–5. [6] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Sixteenth informational supplement. Wayne, PA: CLSI; 2006. [7] Kirby JT, Sader HS, Walsh TR, Jones RN. Antimicrobial susceptibility and epidemiology of a worldwide collection of Chryseobacterium spp.: report from the SENTRY Antimicrobial Surveillance Program (1997–2001). J Clin Microbiol 2004;42:445–8. [8] Arakawa Y, Shibata N, Shibayama K, Kurokawa H, Yagi T, Fujiwara H, et al. Convenient test for screening metallo--lactamase-producing Gram-negative bacteria by using thiol compounds. J Clin Microbiol 2000;38:40–3. [9] Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem–EDTA disk method for differentiation of metallo--lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002;40: 3798–801. [10] Shibata N, Doi Y, Yamane K, Yagi T, Kurokawa H, Shibayama K. PCR typing of genetic determinants for metallo--lactamases and integrases carried by Gram-negative bacteria isolated in Japan, with focus on the class 3 integron. J Clin Microbiol 2003;41:5407–13. [11] Lauretti L, Riccio ML, Mazzariol A, Cornaglia G, Amicosante G, Fontana R, et al. Cloning and characterization of blaVIM , a new integron-borne metallo-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother 1999;43:1584–90. [12] Matsumura N, Minami S, Watanabe Y, Iyobe S, Mitsuhashi S. Role of permeability in the activities of -lactams against Gram-negative bacteria which produce a group 3 -lactamase. Antimicrob Agents Chemother 1999;43: 2084–6. [13] Bellais S, Aubert D, Naas T, Nordmann P. Molecular and biochemical heterogeneity of class B carbapenem-hydrolyzing -lactamases in Chryseobacterium meningosepticum. Antimicrob Agents Chemother 2000;44: 1878–86.
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Heterogeneity of blaIND metallo--lactamase-producing Chryseobacterium indologenes isolates detected in Hefei, China Xiang-hong Lin a , Yuan-hong Xu a,∗ , Jun Cheng b , Tao Li a , Zhong-xin Wang a a b
Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China Department of Infectious Diseases, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
a r t i c l e
i n f o
Article history: Received 19 January 2008 Accepted 27 April 2008 Keywords: Chryseobacterium indologenes Carbapenem Resistance Genotype Metallo--lactamase
a b s t r a c t This study investigated the phenotypic and genetic properties of 28 clinical isolates of metallo-lactamase (MBL)-producing Chryseobacterium indologenes recovered from a university hospital. Twenty isolates were confirmed to carry a blaIND gene. Among them, 9 isolates were confirmed to carry the IND-1 gene, 10 contained the IND-2 gene and 1 had blaIND-3 alleles. One strain (No. 7) confirmed to be carrying the blaIND-1 gene was susceptible to imipenem and was negative in phenotypic methods. The data revealed that the antimicrobial resistance patterns of C. indologenes harbouring MBL genes are remarkably distinct. Two specific types of MBL genes, blaIND-1 and blaIND-2 , were identified to be the major genotype for C. indologenes isolated from Hefei, China. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Over the last few years, interest in Chryseobacterium spp. has grown rapidly due to the emergence of multidrug-resistant Chryseobacterium spp. Chryseobacterium spp. are intrinsically resistant to many -lactams, most commonly associated with the production of high levels of metallo--lactamases (MBLs). Chryseobacterium indologenes is a species commonly reported to cause various clinical syndromes [1]. Production of biofilm on foreign materials as well as protease activity may play an important role in the virulence of invasive infections due to C. indologenes [2]. Chryseobacterium indologenes isolates usually possess MBLs [3], which confer resistance to carbapenems such as imipenem (IPM) and meropenem (MEM). A specific type of MBL, BlaIND, has been identified in isolates of C. indologenes, with more than ten blaIND alleles identified and registered in GenBank. MBL-producing Gram-negative organisms, including Chryseobacterium meningosepticum, have recently been described in China [4,5]. Little is known about the distribution and heterogeneity of blaIND in clinical isolates of C. indologenes. The aim of the present study was to assess the prevalence and types of MBL-positive bacteria among 28 clinical C. indologenes isolated between March 2004 and December 2006 in Hefei, China. Moreover, the phenotypes and
∗ Corresponding author. Tel.: +86 551 292 3175; fax: +86 551 365 8417. E-mail address: [email protected] (Y.-h. Xu).
genetic properties of MBL-producing C. indologenes strains were also determined. 2. Materials and methods 2.1. Bacterial strains and antimicrobial susceptibility testing Twenty-eight clinically isolated C. indologenes were collected between March 2004 and December 2006 at the First Affiliated Hospital of Anhui Medical University, a 1500-bed public tertiary care teaching hospital in Hefei, China. All isolates were nonduplicates and were re-identified using MicroScan WA40 (Dade Behring Corp., Deerfield, IL). Minimum inhibitory concentration determination of antimicrobial agents was performed by the agar dilution method according to the Clinical and Laboratory Standards Institute [6]. Selected drugs active against Gram-positive bacteria were tested against C. indologenes and the results were interpreted according to breakpoints approved for staphylococci [7]. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as quality control strains. 2.2. Phenotypic detection of MBLs Two phenotypic detection methods, the three-dimensional (3D) test and the IPM–ethylene diamine tetraacetic acid (EDTA) (2mercaptopropionic acid) disc synergy test, were designed to screen for MBLs. For the 3D tests, crude enzyme extracts were prepared
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.04.028
X.-h. Lin et al. / International Journal of Antimicrobial Agents 32 (2008) 398–400
from isolates by cell sonication. Crude enzyme extract only (30 L), enzyme extract (30 L) plus 2 L of 2-mercaptopropionic acid and enzyme extract (30 L) plus 5 L of 0.5 M EDTA (pH 8.0) were each loaded in slots of a Mueller–Hinton agar plate. One IPM disc (10 g) (Oxoid, Basingstoke, UK) was placed in the centre of the plate. Escherichia coli ATCC 25922 was inoculated as the indicator isolate. Additionally, screening for MBLs was performed by the disc approximation test using a modification of the procedure described by Arakawa et al. [8] and Yong et al. [9]. Three IPM discs were placed 30 mm apart (centre to centre) on a Mueller–Hinton agar plate, onto which C. indologenes was spread. One IPM disc contained 5 L of 0.5 M EDTA (pH 8.0) and one contained 2 L of 2-mercaptopropionic acid. Pseudomonas aeruginosa 119/05 (VIM-2 producer) was used as a positive control. 2.3. Polymerase chain reaction (PCR) amplification and DNA sequencing PCR analysis was performed on all isolates to confirm the presence of MBL genes. blaIMP-1 , blaVIM-1 , blaVIM-2 , blaSPM-1 and blaIND genes were assayed using previously reported primers [3,10]. Four pairs of primers that amplify the whole blaIND gene were also used (Table 1). Nucleotide automated sequencing of both strands of the PCR products derived with primers that amplify the whole blaIND gene was performed with an ABI Prism 3730 DNA sequencer (Perkin-Elmer, Applied Biosystems Division, Foster City, CA). 2.4. Conjugation, plasmid preparation, ˇ-lactamase assays and isoelectric focusing Conjugation experiments were carried out with streptomycinresistant E. coli C600 strains as recipients. Transconjugants were selected on Mueller–Hinton agar plates containing 100 g/mL streptomycin and 2 g/mL IPM. Plasmid DNA of C. indologenes isolates producing MBL was extracted using QIAquick Gel Extraction Kit (QIAGEN GmbH, Hilden, Germany) and was used as the template DNA in PCR for the detection of blaIND . Carbapenemase activity in cell sonicates from overnight Luria–Bertani broth cultures was determined using a spectrophotometer (UV-SP752; Spectrum Corp., Shanghai, China) as described previously [11]. Inhibition by EDTA was assayed as described previously [11]. The protein content of the sonicates was determined using a commercial assay reagent (Bio-Rad, Foster City, CA) with bovine serum albumin as a standard. Determination of the isoelectric point (pI) of -lactamases was performed in pre-cast 5% polyacrylamide gels containing ampholytes (pH range 3.5–9.5; Ampholine PAGplate; Amersham Biosciences, Uppsala, Sweden) using nitrocefin chromogenic substrate for zymographic detection of -lactamases, as described Table 1 Polymerase chain reaction (PCR) primers used in analysis of the blaIND gene Primer
Sequence (5 –3 )
Amplicon (bp)
Annealing (◦ C)
blaIND Univ
F-GCCCAGGTTAAAGATTTTGTAAT R-CATGGCCACCGCCTTTCCATTC F-ATGAAAAAAAGCATCCG R-CTATTTTTTATTCAGAAGTTC F-ATGAAAAAAAGTATTCAGCTTTTG R-TTATTCCGGCTTTTTATTCTTATC F-ATGAAAAAAAGAATTCAGTTC R-TTATTTTTTGTTAAGAAGTTC F-ATGAGGAAAAATGTTAGGA R-CTACTGTTTTTGTTGATCTAA
580
52
720
48
732
49
720
48
720
49
blaIND1 group blaIND2 group blaIND3 group blaIND4 group
399
previously [11]. TEM-1 (pI 5.4), SHV-1 (pI 7.6), SHV-5 (pI 8.2) and AmpC (pI 8.8) were used as controls.
3. Results and discussion In the antimicrobial susceptibility test, most of the 28 C. indologenes strains were resistant to piperacillin, IPM, MEM, cefotaxime, aztreonam and ceftazidime, showing resistance rates >50% (Table 2). Significantly, 23 (82%) were resistant to IPM and MEM, which are widely used to treat infections caused by multidrugresistant Gram-negative bacteria. Isolates in this study show greater susceptibility to levofloxacin (71%), ciprofloxacin (68%) and gatifloxacin (75%). Moreover, vancomycin and rifampicin, which are more frequently used for Gram-positive bacteria, are an even better choice. Twenty C. indologenes isolates were confirmed to carry a blaIND gene, including 9 isolates carrying blaIND-1 , 10 carrying blaIND-2 and 1 carrying blaIND-3 alleles (Table 2). Among them, eight strains were isolated from respiratory tract specimens, four were isolated from blood, three were from urine and the remaining five were isolated from various other specimens. None of the isolates had other MBL genes except for blaIND . The majority of blaIND -producers isolated from this area had either blaIND-1 or blaIND-2 . IND-2 shared 80% amino acid identity with IND-1 and had a very similar broadspectrum resistance profile, and they were classified in functional subgroup 3a of class B carbapenem-hydrolysing -lactamases [3]. In the current study, two isolates, Nos. 10 and 16, harboured blaIND-1 and blaIND-2 , respectively. Their gene sequences contained mutations and were identified to be two new blaIND alleles, which were registered and deposited in GenBank under accession numbers EF394436 and EF394439. Interestingly, of the 20 blaIND -carrying isolates, 19 were found to be positive in two phenotypic MBL detection methods. One C. indologenes isolate (No. 7) gave a negative result both with the 3D test and the disc synergy test. Overall, the sensitivity and specificity of the results in different methods were 100% and 95%, respectively. Nineteen (83%) of the 23 IPM-resistant isolates harboured a blaIND gene. However, repeated attempts to transfer the MBL gene by conjugation failed. Plasmids were extracted from three C. indologenes strains producing MBLs but none of them harboured MBL genes. The results indicate that blaIND genes are very likely located on the chromosome. With the exception of isolate No. 7, cell sonicates of all blaIND positive isolates hydrolysed IPM in spectrophotometric assays and this activity was inhibited by EDTA. Using extracts of four C. indologenes strains randomly, the mean IPM-hydrolysing activity was ca. 70 mol/min/mg of protein. Meanwhile, isoelectric focusing of 19 blaIND -positive isolate extracts (not including No. 7) detected a -lactamase with a pI of 8.6–8.8, in keeping with IND-type lactamase. Other pI values could not be detected. The naturally occurring resistance to many antimicrobial agents such as aztreonam may not be linked to other -lactamases in C. indologenes. Although four isolates were highly resistant to IPM in the current study, we failed to amplify blaIND , which may indicate that other carbapenem resistance mechanisms are present in C. indologenes isolates. On the other hand, isolate No. 7 was confirmed to be carrying blaIND-1 but was susceptible to IPM and MEM. We found no phenotypic evidence of MBL production for this strain. This phenomenon of low-level resistance to carbapenems for other MBL-producing Gram-negative bacteria was confirmed, as previously described [12]. The variation in the permeability coefficient for each -lactam may play a major role. Further investigations for C. indologenes No. 7 are necessary to evaluate the role of permeability coefficient changes.
400
X.-h. Lin et al. / International Journal of Antimicrobial Agents 32 (2008) 398–400
Table 2 Minimum inhibitory concentrations (MICs) of antibiotics for Chryseobacterium indologenes isolates with various metallo--lactamase (MBL) genes No.
Origin
MBL
1 3 4 6 7 9 10 12 14 16 17 18 19 20 22 24 25 26 27 28
Urine Blood Sputum Sputum Blood Pus Sputum Sputum Stool Wound Sputum Wound Sputum Urine Blood Sputum Sputum Blood Urine Pus
blaIND-1 blaIND-2 blaIND-2 blaIND-2 blaIND-1 blaIND-2 blaIND-1 blaIND-1 blaIND-1 blaIND-2 blaIND-2 blaIND-1 blaIND-2 blaIND-1 blaIND-2 blaIND-2 blaIND-3 blaIND-1 blaIND-2 blaIND-1
MIC (mg/L) PIP
CAZ
CIP
GAT
ATM
IPM
MEM
LVX
CTX
TZP
RIF
VAN
128 >128 64 >128 32 >128 >128 64 128 >128 >128 >128 64 >128 128 >128 >128 128 64 >128
64 >128 32 >128 8 >128 128 128 64 32 >128 16 4 >128 >128 128 >128 32 16 64
0.25 4 0.25 2 0.25 8 16 2 4 0.5 >32 0.5 0.25 2 4 16 8 >32 0.25 0.5
1 2 0.5 4 1 32 8 0.5 2 1 8 0.5 1 8 16 4 8 16 0.5 1
>128 128 32 >128 16 >128 >128 128 128 64 >128 128 32 128 >128 128 >128 64 64 128
32 128 64 128 4 64 >128 32 64 64 >128 64 32 >128 128 64 128 128 32 64
64 >128 64 128 4 32 128 32 32 128 >128 32 32 >128 128 32 64 >128 64 64
2 16 1 4 0.5
>128 128 32 4 8 32 16 4 32 128 >128 64 64 >128 128 64 128 >128 16 32
8 32 4 16 8 32 64 4 8 32 >128 2 4 64 8 8 16 4 4 8
0.5 2 1 32 4 4 16 2 4 16 64 2 2 4 8 8 4 64 1 2
4 4 2 64 16 64 128 8 8 32 128 4 8 32 64 64 16 32 2 4
8 1 2 1 32 0.5 1 1 2 8 2 4 1 0.5
PIP, piperacillin; CAZ, ceftazidime; CIP, ciprofloxacin; GAT, gatifloxacin; ATM, aztreonam; IPM, imipenem; MEM, meropenem; LVX, levofloxacin; CTX, cefotaxime; TZP, piperacillin–tazobactam; RIF, rifampicin; VAN, vancomycin.
BlaIND, like BlaB and GOB from C. meningosepticum, confirmed the variability of chromosome-located class B -lactamases [3,13]. Chryseobacterium meningosepticum expresses two non-related MBLs, BlaB and GOB, with genetic and biochemical heterogeneity. In contrast to C. meningosepticum, C. indologenes may possess only one type of MBL, BlaIND. Although the prevalence of C. indologenes is low, it seems clear that the proportion of MBL-producing and multidrug-resistant isolates in our collection was higher than has been reported for C. meningosepticum in other parts of the world [4,7]. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Hsueh PR, Teng LJ, Yang PC, Ho SW, Hsieh WC, Luh KT. Increased incidence of nosocomial Chryseobacterium indologenes infections in Taiwan. Eur J Clin Microbiol Infect Dis 1997;16:568–74. [2] Pan HJ, Teng LJ, Chen YC, Hsueh PR, Yang PC, Ho SW, et al. High protease activity of Chryseobacterium indologenes isolates associated with invasive infection. J Microbiol Immunol Infect 2000;33:223–6. [3] Bellais S, Poirel L, Leotard S, Naas T, Nordmann P. Genetic diversity of carbapenem-hydrolyzing metallo--lactamases from Chryseobacterium (Flavobacterium) indologenes. Antimicrob Agents Chemother 2000;44:3028–34. [4] Chen GX, Zhang R, Zhou HW. Heterogeneity of metallo-beta-lactamases in clinical isolates of Chryseobacterium meningosepticum from Hangzhou, China. J Antimicrob Chemother 2006;57:750–2.
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