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July-September 2014
Correspondences
349
Prevalence and molecular characterisation of metallo‑beta‑lactamase producing strains of imipenem‑resistant Pseudomonas aeruginosa in Turkey Dear Editor, Acquired carbapenem resistance due to the production of metallo beta‑lactamases (MBLs) has been increasingly reported, particularly for Pseudomonas aeruginosa and Acinetobacter spp. Prompt detection and recognition of the MBLs is important to implement adequate counter‑measures to control the spread of the organisms bearing these enzymes, and proper treatment of infections caused by MBL‑producing microorganisms. The aim of this study is to identify imipenem (IMP) resistant strains of P. aeruginosa from various clinical specimens of inpatients at our hospital and to investigate the presence of MBL‑producing strains among these isolates by phenotypic and genotypic methods and to compare the results with each other. The study included randomly selected un‑duplicate 38 IMP resistant P. aeruginosa isolates from clinical specimens collected in 2010. Isolates were identified by conventional testing and VITEK‑2 (bioMerieux, France) automatic identification system. The IMP susceptibility testing was performed by the E‑Test (AB Biodisk, Solna, Sweden) according to manufacturer’s recommendations. P. aeruginosa ATCC 27853 was used as a control. MBL production of IMP resistant isolates was screened phenotypically by the double disk synergy test (DDST) using an IMP disk (10 µg) and an IMP‑EDTA disk (750 µg) on Mueller-Hinton agar (Salubris, Turkey) with 10 mm distance from the edge to the edge of the disk. After overnight incubation, the presence of even a synergistic inhibition zone was interpreted as positive.[1] IMP‑EDTA disks were prepared from commercially available chemicals with 0.5 M EDTA (Sigma Chemical, Germany). Multiplex PCR and subsequent sequencing were carried on in order to identify the blaVIM and blaIMP MBL genes.[2] PCR amplicons were identified by multiplex PCR were sequenced. A total of 38 IMP resistant P. aeruginosa strains were analysed. Strains were dominantly isolated from the pus (n: 16, 42.1%), also blood cultures (n: 10, 26.3%), urinary tract (n: 6, 15.8%) and tracheal aspirate specimens (n: 6, 15.8%). Bacterial strains were isolated mainly from intensive care unit (n: 18, 47%), general surgery (n: 12, 32%) and internal medicine (n: 8, 21%). The majority of MBL‑producing isolates were isolated from pus and from intensive care unit. MIC90 values for IMP was found to be 32 µg/ml. Carbapenem resistant isolates were detected concurrently resistant to ciprofloxacin 82%, gentamicin 91% and amikacin 82%. All strains were susceptible to colistin and polimyxin B (MIC90 values were detected, 0.50 µg/ml for both). The MBL enzyme was found positive in 27 of 38 (71.05%) IMP resistant P. aeruginosa
isolates by DDST. Eight of 38 (21.05%) MBL‑positive isolates were confirmed to be positive for MBL by PCR [Figure 1]. Nucleotide sequence analysis of the amplicons showed seven strains positive for the blaVIM‑2, one strain positive for the blaIMP‑9 gene, which was confirmed by BLAST. No other MBLs were detected. Although 20 false‑positive results were found by the MBL‑DDST, there was no false‑negative result among the isolates of P. aeruginosa [Table 1]. The occurrence of an MBL‑positive isolate in a hospital setting poses a therapeutic problem, as well as a serious concern for infection control management. The prevalence of MBL‑positive strains among IPM resistant P. aeruginosa isolates from our hospital was estimated at 71.05% (27/38) by DDST and 21% (8/38) by PCR. The prevalence of P. aeruginosa that produce MBL can be markedly different in distinct geographical areas, even among different hospitals in the same area. Our results in this study remind us that metallo enzymes have become a serious clinical and therapeutic problem for our hospital.
Figure 1: Detection of MBL genes (blaVIM and blaIMP) by multiplex PCR. 1. Lane (1-16); M, 4-4, 12-12, A-1, 5-b, 16-16, 1-H, A-9, 8-b, 3-3, 3-b, 18-18, A-4, A-12, A-6, 10-10, 2. Lane (1-4); M, 19-19, A-17, A-20. BlaVIM genes (261 bp): 16-16, 8-b, 3-3, 18-18, A-12, A-6, A-17, BlaIMP gene (765 bp): 1-H
Table 1: PCR positivity among DDSTa positive and negative strains of P. aeruginosa MBL‑DDST(+) MBL‑DDST(‑) Total PCR (+) 8 0 8 PCR (‑) 20 10 30 Total 28 10 38 DDST: Double disk synergy test
a
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350
Indian Journal of Medical Microbiology
The accurate identification and reporting of MBL‑producing P. aeruginosa will aid infection control practitioners in preventing the spread of these multidrug‑resistant isolates. Many phenotyping methods have performed to search MBL enzymes of P. aeruginosa strains. All these methods are based on the ability of metal chelators, such as EDTA and thiol‑based compounds, to inhibit the activity of MBL. The DDST has been reported to be simple, inexpensive phenotypic resources for detection of MBL that could be easily incorporated into the routines of clinical laboratories.[1,3] Although there was any false‑negative results were detected, 20 false‑positive results were found by the MBL‑DDST [Table 1]. Chu et al.,[3] reported that the false positive results in P. aeruginosa by E‑test and disk synergy methods were due to the EDTA susceptibility of the strains. It is known that EDTA may increase bacterial cell wall permeability and that zinc (chelated by EDTA) accelerates IPM decomposition and decreases OprD expression of P. aeruginosa.[4] In this study, not all MBL genes only blaVIM and blaIMP MBL genes were invesitigated. The reason for false positive result in P. aeruginosa by DDTS could be due to this reason. We conclude that synergy‑based test results should be confirmed further genetically until standard criteria for performing and evaluating the phenotypic MBL‑screening tests are established for EDTA‑susceptible and ‑non‑susceptible strains.
vol. 32, No. 3
for differentiating metallo‑beta‑lactamase‑producing isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2003;41:4623‑9. 2. Ohara M, Kouda S, Onodera M, Fujiue M, Sasaki M, Kohara T, et al. Molecular Characterization of imipenem‑resistant Pseudomonas aeruginosa in Hiroshima, Japan. Microbiol Immunol 2007;51:271‑7. 3. Chu YW, Cheung TK, Ngan JY, Kam KM. EDTA susceptibility leading to false detection of metallo‑beta‑lactamase in Pseudomonas aeruginosa by E‑test and an imipenem‑EDTA disk method. Int J Antimicrob Agents 2005;26:340‑1. 4. Qu TT, Zhang JL, Wang J, Tao J, Yu YS, Chen YG, et al. Evaluation of phenotypic tests for detection of metallo‑β‑lactamase‑producing Pseudomonas aeruginosa strains in China. J Clin Microbiol 2009;47:1136‑42.
* N Ozkalay Yilmaz, N Agus, E Bozcal, A Uzel Department of Microbiology Laboratory (NOY, NA), Tepecik Educational and Research Hospital, Department of Biology (EB, AU), Basic and Industrial Microbiology Section, Faculty of Science, Ege University, Izmir, Turkey *Corresponding author (email: ) Received: 23‑05‑2013 Accepted: 10‑08‑2013
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Acknowledgments
Quick Response Code:
This work was partially funded by a grant from the Ege University Scientific Research Foundation Grant No: 2009 Fen 017.
Website: www.ijmm.org PMID: ***
References
DOI: 10.4103/0255-0857.136622
1. Lee K, Lim YS, Yong D, Yum JH, Chong Y. Evaluation of the Hodge test and the imipenem‑EDTA double‑disk synergy test
Rejection of a manuscript: The other side of the story Dear Editor, We have read the article “Rejection of a manuscript” by Kapil, with interest.[1] The points raised by this article are relevant in the field of microbiology as well as in other disciplines. As mentioned in the article, the reviewers are indeed the pillars of the entire process. In view of today’s motto of “publish or perish” many budding authors are encountered daily.[2] Surprisingly, the number of scientific journals is also mushrooming in a proportionate manner.
For the first timer authors, getting a manuscript rejected is an entire novel experience.[3] For many of them, it results in heartburns. The role of an experienced reviewer in this matter lies in giving constructive comments, and suggestions to improve the manuscript. The time taken to review also should be reasonable. Many reviewers will be busy with their routine work besides reviewing the manuscripts. So, if they could not give adequate time to review an article, they should intimate the same to the journal website promptly. Many of the articles submitted to the journal would not meet the criteria set by that particular
www.ijmm.org
Copyright of Indian Journal of Medical Microbiology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
July-September 2014
Correspondences
349
Prevalence and molecular characterisation of metallo‑beta‑lactamase producing strains of imipenem‑resistant Pseudomonas aeruginosa in Turkey Dear Editor, Acquired carbapenem resistance due to the production of metallo beta‑lactamases (MBLs) has been increasingly reported, particularly for Pseudomonas aeruginosa and Acinetobacter spp. Prompt detection and recognition of the MBLs is important to implement adequate counter‑measures to control the spread of the organisms bearing these enzymes, and proper treatment of infections caused by MBL‑producing microorganisms. The aim of this study is to identify imipenem (IMP) resistant strains of P. aeruginosa from various clinical specimens of inpatients at our hospital and to investigate the presence of MBL‑producing strains among these isolates by phenotypic and genotypic methods and to compare the results with each other. The study included randomly selected un‑duplicate 38 IMP resistant P. aeruginosa isolates from clinical specimens collected in 2010. Isolates were identified by conventional testing and VITEK‑2 (bioMerieux, France) automatic identification system. The IMP susceptibility testing was performed by the E‑Test (AB Biodisk, Solna, Sweden) according to manufacturer’s recommendations. P. aeruginosa ATCC 27853 was used as a control. MBL production of IMP resistant isolates was screened phenotypically by the double disk synergy test (DDST) using an IMP disk (10 µg) and an IMP‑EDTA disk (750 µg) on Mueller-Hinton agar (Salubris, Turkey) with 10 mm distance from the edge to the edge of the disk. After overnight incubation, the presence of even a synergistic inhibition zone was interpreted as positive.[1] IMP‑EDTA disks were prepared from commercially available chemicals with 0.5 M EDTA (Sigma Chemical, Germany). Multiplex PCR and subsequent sequencing were carried on in order to identify the blaVIM and blaIMP MBL genes.[2] PCR amplicons were identified by multiplex PCR were sequenced. A total of 38 IMP resistant P. aeruginosa strains were analysed. Strains were dominantly isolated from the pus (n: 16, 42.1%), also blood cultures (n: 10, 26.3%), urinary tract (n: 6, 15.8%) and tracheal aspirate specimens (n: 6, 15.8%). Bacterial strains were isolated mainly from intensive care unit (n: 18, 47%), general surgery (n: 12, 32%) and internal medicine (n: 8, 21%). The majority of MBL‑producing isolates were isolated from pus and from intensive care unit. MIC90 values for IMP was found to be 32 µg/ml. Carbapenem resistant isolates were detected concurrently resistant to ciprofloxacin 82%, gentamicin 91% and amikacin 82%. All strains were susceptible to colistin and polimyxin B (MIC90 values were detected, 0.50 µg/ml for both). The MBL enzyme was found positive in 27 of 38 (71.05%) IMP resistant P. aeruginosa
isolates by DDST. Eight of 38 (21.05%) MBL‑positive isolates were confirmed to be positive for MBL by PCR [Figure 1]. Nucleotide sequence analysis of the amplicons showed seven strains positive for the blaVIM‑2, one strain positive for the blaIMP‑9 gene, which was confirmed by BLAST. No other MBLs were detected. Although 20 false‑positive results were found by the MBL‑DDST, there was no false‑negative result among the isolates of P. aeruginosa [Table 1]. The occurrence of an MBL‑positive isolate in a hospital setting poses a therapeutic problem, as well as a serious concern for infection control management. The prevalence of MBL‑positive strains among IPM resistant P. aeruginosa isolates from our hospital was estimated at 71.05% (27/38) by DDST and 21% (8/38) by PCR. The prevalence of P. aeruginosa that produce MBL can be markedly different in distinct geographical areas, even among different hospitals in the same area. Our results in this study remind us that metallo enzymes have become a serious clinical and therapeutic problem for our hospital.
Figure 1: Detection of MBL genes (blaVIM and blaIMP) by multiplex PCR. 1. Lane (1-16); M, 4-4, 12-12, A-1, 5-b, 16-16, 1-H, A-9, 8-b, 3-3, 3-b, 18-18, A-4, A-12, A-6, 10-10, 2. Lane (1-4); M, 19-19, A-17, A-20. BlaVIM genes (261 bp): 16-16, 8-b, 3-3, 18-18, A-12, A-6, A-17, BlaIMP gene (765 bp): 1-H
Table 1: PCR positivity among DDSTa positive and negative strains of P. aeruginosa MBL‑DDST(+) MBL‑DDST(‑) Total PCR (+) 8 0 8 PCR (‑) 20 10 30 Total 28 10 38 DDST: Double disk synergy test
a
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[Downloaded free from http://www.ijmm.org on Saturday, July 12, 2014, IP: 220.225.230.107] || Click here to download free Android application for this journal
350
Indian Journal of Medical Microbiology
The accurate identification and reporting of MBL‑producing P. aeruginosa will aid infection control practitioners in preventing the spread of these multidrug‑resistant isolates. Many phenotyping methods have performed to search MBL enzymes of P. aeruginosa strains. All these methods are based on the ability of metal chelators, such as EDTA and thiol‑based compounds, to inhibit the activity of MBL. The DDST has been reported to be simple, inexpensive phenotypic resources for detection of MBL that could be easily incorporated into the routines of clinical laboratories.[1,3] Although there was any false‑negative results were detected, 20 false‑positive results were found by the MBL‑DDST [Table 1]. Chu et al.,[3] reported that the false positive results in P. aeruginosa by E‑test and disk synergy methods were due to the EDTA susceptibility of the strains. It is known that EDTA may increase bacterial cell wall permeability and that zinc (chelated by EDTA) accelerates IPM decomposition and decreases OprD expression of P. aeruginosa.[4] In this study, not all MBL genes only blaVIM and blaIMP MBL genes were invesitigated. The reason for false positive result in P. aeruginosa by DDTS could be due to this reason. We conclude that synergy‑based test results should be confirmed further genetically until standard criteria for performing and evaluating the phenotypic MBL‑screening tests are established for EDTA‑susceptible and ‑non‑susceptible strains.
vol. 32, No. 3
for differentiating metallo‑beta‑lactamase‑producing isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2003;41:4623‑9. 2. Ohara M, Kouda S, Onodera M, Fujiue M, Sasaki M, Kohara T, et al. Molecular Characterization of imipenem‑resistant Pseudomonas aeruginosa in Hiroshima, Japan. Microbiol Immunol 2007;51:271‑7. 3. Chu YW, Cheung TK, Ngan JY, Kam KM. EDTA susceptibility leading to false detection of metallo‑beta‑lactamase in Pseudomonas aeruginosa by E‑test and an imipenem‑EDTA disk method. Int J Antimicrob Agents 2005;26:340‑1. 4. Qu TT, Zhang JL, Wang J, Tao J, Yu YS, Chen YG, et al. Evaluation of phenotypic tests for detection of metallo‑β‑lactamase‑producing Pseudomonas aeruginosa strains in China. J Clin Microbiol 2009;47:1136‑42.
* N Ozkalay Yilmaz, N Agus, E Bozcal, A Uzel Department of Microbiology Laboratory (NOY, NA), Tepecik Educational and Research Hospital, Department of Biology (EB, AU), Basic and Industrial Microbiology Section, Faculty of Science, Ege University, Izmir, Turkey *Corresponding author (email: ) Received: 23‑05‑2013 Accepted: 10‑08‑2013
Access this article online
Acknowledgments
Quick Response Code:
This work was partially funded by a grant from the Ege University Scientific Research Foundation Grant No: 2009 Fen 017.
Website: www.ijmm.org PMID: ***
References
DOI: 10.4103/0255-0857.136622
1. Lee K, Lim YS, Yong D, Yum JH, Chong Y. Evaluation of the Hodge test and the imipenem‑EDTA double‑disk synergy test
Rejection of a manuscript: The other side of the story Dear Editor, We have read the article “Rejection of a manuscript” by Kapil, with interest.[1] The points raised by this article are relevant in the field of microbiology as well as in other disciplines. As mentioned in the article, the reviewers are indeed the pillars of the entire process. In view of today’s motto of “publish or perish” many budding authors are encountered daily.[2] Surprisingly, the number of scientific journals is also mushrooming in a proportionate manner.
For the first timer authors, getting a manuscript rejected is an entire novel experience.[3] For many of them, it results in heartburns. The role of an experienced reviewer in this matter lies in giving constructive comments, and suggestions to improve the manuscript. The time taken to review also should be reasonable. Many reviewers will be busy with their routine work besides reviewing the manuscripts. So, if they could not give adequate time to review an article, they should intimate the same to the journal website promptly. Many of the articles submitted to the journal would not meet the criteria set by that particular
www.ijmm.org
Copyright of Indian Journal of Medical Microbiology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
