|Year : 2013 | Volume
| Issue : 3 | Page : 257-260
Co-existence of Pseudomonas-derived cephalosporinase among plasmid encoded CMY-2 harbouring isolates of Pseudomonas aeruginosa in north India
S Upadhyay1, S Mishra1, MR Sen1, T Banerjee1, A Bhattacharjee2
1 Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Microbiology, Assam University, Silchar, Assam, India
|Date of Submission||04-Feb-2013|
|Date of Acceptance||26-May-2013|
|Date of Web Publication||25-Jul-2013|
M R Sen
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh
Source of Support: Department of Biotechnology (Grant BT/
PR11812/BRB/10/692), Government of India. BHU Project P.07.467., Conflict of Interest: None
Context: In Pseudomonas aeruginosa, AmpC β-lactamases are often responsible for high-level resistance to β-lactam antibiotics. The co-production of plasmid-mediated AmpC along with chromosomal Pseudomonas-derived cephalosporinases thus remain a serious clinical concern owing to high resistance spectrum towards antibiotics. Aim: The present study was performed to investigate the co-existence of both chromosomally-encoded and plasmid-mediated AmpC β-lactamase among clinical isolates of P. aeruginosa. Setting and Design: It is a cross-sectional study carried out in the Department of Microbiology in a tertiary referral hospital of northern India. Methods and Methods: A total of 329 consecutive, non-duplicate clinical isolates of P. aeruginosa, were selected for the detection of AmpC β-lactamases and confirmed for AmpC production by modified three dimensional (M3D) test. Ceftazidime -imipenem antagonism test was used to detect inducible AmpC producers. Molecular characterisation of chromosomally-encoded blaPDC and plasmid-mediated AmpC gene was studied by performing polymerase chain reaction (PCR). Result: A total of 214 (65%) isolates were confirmed for AmpC production by M3D test. On performing multiplex PCR, 27 isolates were detected posessing blaCMY type of plasmid-mediated AmpC gene. While 48 isolates were found to harbour chromosomally-encoded blaPDC gene co-production of both chromosomal and plasmid-encoded AmpC was reported in eleven isolates. Conclusions: Although these chromosomally-encoded cephalosporinases might spread more slowly than mobilised AmpC, but it is likely that in the present scenario of intense antibiotic pressure, this will become an increasing problem and may further limit our antibiotic choices.
Keywords: AmpC β-lactamases, chromosomal AmpC, Pseudomonas?derived cephalosporinase-lactamases, chromosomal AmpC, Pseudomonas-derived cephalosporinase
|How to cite this article:|
Upadhyay S, Mishra S, Sen M R, Banerjee T, Bhattacharjee A. Co-existence of Pseudomonas-derived cephalosporinase among plasmid encoded CMY-2 harbouring isolates of Pseudomonas aeruginosa in north India. Indian J Med Microbiol 2013;31:257-60
|How to cite this URL:|
Upadhyay S, Mishra S, Sen M R, Banerjee T, Bhattacharjee A. Co-existence of Pseudomonas-derived cephalosporinase among plasmid encoded CMY-2 harbouring isolates of Pseudomonas aeruginosa in north India. Indian J Med Microbiol [serial online] 2013 [cited 2020 May 30];31:257-60. Available from: http://www.ijmm.org/text.asp?2013/31/3/257/115629
| ~ Introduction|| |
Pseudomonas aeruginosa is one of the major etiological agents of nosocomial infections; it has at least three ampD genes, enhanced AmpC production occurs in a stepwise fashion.  The most common resistance mechanism against various β-lactam drugs is the selection of mutations leading to the hyperproduction of chromosomal AmpC. , The derepressed mutants can be selected in clinical settings expressing resistant phenotype. Several such chromosomally mediated Pseudomonas-derived cephalosporinases (PDC) with extended-spectrum cephalosporinases activities have been reported among P. aeruginosa. In addition to chromosomal AmpC, the production of plasmid-mediated AmpC represents a new threat in the treatment of infection caused by P. aeruginosa.
| ~ Materials and Methods|| |
The aim of this study was to characterise the chromosomal as well as plasmid-encoded AmpC β-lactamase among clinical isolates of P. aeruginosa in a tertiary referral hospital in northern India.
Strain collection and phenotypic microbiological methods
In this study, a total of 329 non repeat, consecutive clinical isolates of P. aeruginosa were isolated from inpatient (n0 = 256) and outpatient (n = 73) departments from April 2008 to September 2009. Written informed consent was obtained from the patient (s) for publication of this manuscript and accompanying images. This work has been ethically approved by the chairperson of the ethical committee of the Institute. The isolates were identified by conventional methods.  A previously confirmed clinical isolate of Escherichia coli harbouring blaCMY-2 type AmpC was taken as positive control and E. coli American Type Culture Collection (ATCC) 25922 as negative control. Minimum Inhibitory Concentration (MIC) of all the isolates were carried out by agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) recommendation  with cefotaxime, ceftazidime, ceftriaxone (Hi Media, Mumbai, India), cefepime (Alembic Ltd, Vadodra, India), aztreonam (Aristo Pharmaceuticals Ltd., Mumbai, India), imipenem (United Biotech, Solan, India) and meropenem (Astra Zeneca Pharmaceuticals Ltd, Bangalore, India).
Isolates were tested and confirmed for AmpC production by Ceftazidime-imipenem antagonism test (CIAT) (for inducible/chromosomal AmpC detection)  and modified three-dimensional (M3D) test as described previously.  The Metallo β-lactamase (MBL) status of the strains was established by the imipenem-ethylene diamine tetra-acetic acid (EDTA) method. 
Genotypic detection of chromosomally encoded and plasmid mediated AmpC gene
All the AmpC producing isolates were subjected to genotypic characterisation for detection of inducible/chromosomal PDC gene and for different family of plasmid-mediated AmpC gene by multiplex polymerase chain reaction (PCR) as described previously.  For characterisation of chromosomal AmpC gene, primers (IDT, USA) specific for blaPDC genes were used for reaction with bacterial DNA as template. 
Sequencing analysis of plasmid mediated AmpC gene family
To identify the AmpC gene type detected in the multiplex PCR assay of different family of plasmid encoded AmpC gene, DNA sequence analyses of the amplicons was performed. Amplified PCR products were purified and subjected to DNA sequencing (Genei, Bangalore). Each sequence was then compared with already known β-lactamase gene sequences by using the Basic Local Alignment Search Tool (BLAST) suite of program.
| ~ Results|| |
Among the test isolates, 214 (65%) were detected as AmpC positive by M3D test, of which 71 (33.1%) were suspected for inducible AmpC production by CIAT [Figure 1]. Majority the AmpC positive isolates were obtained from hospitalized patients (n0 = 203), others were from outpatients department (n = 11). On performing PCR for the presence of chromosomal AmpC gene, 48 isolates of P. aeruginosa harboured PDC gene [Figure 2] while plasmid-mediated CIT family of AmpC gene was present in total 27 isolates, of which 11 isolates were harbouring both chromosomal as well as plasmid-mediated AmpC gene (i.e., blaPDC and blaCIT ). Sequencing analysis of the PCR product of CIT family of AmpC gene revealed that sequenced isolates harboured CMY-2 variant gene.
Out of 71 inducible AmpC producers detected phenotypically, presence of chromosomal PDC gene was demonstrated in 30 (42.5%) isolates while among remaining 18 isolates, 14 were phenotypically co-producing metallo β-lactamase enzyme [Table 1] and [Figure 3].
|Table 1: Distribution of chromosomal and plasmid encoded AmpC β-lactamase genes among non-fermenters |
Click here to view
|Figure 1: Ceftazidime-imipenem antagonism test: Positive test showing flattening of zone of ceftazidime towards imipenem|
Click here to view
|Figure 2: Agarose gel showing PDC gene. Lane M showing the 100 bp DNA ladder; lane 1,2,3,5,6 and 7 showing PDC (1243 bp); lane 4, genotypically negative|
Click here to view
|Figure 3: Co-production of MBL among inducible AmpC positive P. aeruginosa|
Click here to view
Strains harbouring chromosomal and plasmidic AmpC gene (i.e., n0 = 64) were predominantly cultured from pus ( n = 26), urine ( n = 13), burn wound ( n = 09), blood ( n = 06), endotracheal tube (ETT) aspirates ( n = 07) and other body sites/fluids ( n = 03).
MIC study showed that isolates harbouring only chromosomal AmpC gene had MIC value in the susceptible to intermediate range for all the cephalosporins and aztreonam tested while isolates harbouring both chromosomal and plasmid-mediated AmpC, MIC values for cephalosporins and aztreonam was in the resistant range. However, in both cases, carbapenem MICs were in the susceptible range while for isolates coproducing AmpC and MBL, MIC for all the β lactams were in the resistant range [Table 2].
|Table 2: MIC of blaPDC harbouring isolates against different β-lactam drugs|
Click here to view
| ~ Discussion|| |
In the present study, besides plasmid-mediated AmpC β-lactamase, variants of the chromosomally-mediated AmpC enzyme PDC, have also been observed among P. aeruginosa. Another study from France reported 10 variants of PDC (PDC 1-10) gene, in which several variants showed reduced susceptibility for ceftazidime, cefepime and imipenem, thus demonstrating an extended spectrum AmpC β-lactamase property.  Reduced susceptibility was observed with recombinant P. aeruginosa strains expressing an AmpC β-lactamase that had an alanine residue at position 105.  In the present study, unlike previous one, strains harbouring PDC gene did not constitute any background for carbapenem resistance. However, when expressed alone or along with plasmidic AmpC blaCMY-2 , they showed intermediate to resistant profile against 3 rd , 4 th generation cephalosporins and monobactam. Thus the presence of AmpC gene with different resistance mechanism offers the increased risk of extended spectrum AmpC β-lactamase in this region.
The CIAT was able to detect maximum number of blaPDC harbouring isolates. However, isolates producing PDC along with MBL could not be detected by the same method. This could be due to hydrolysis of imipenem by MBL, another resistance mechanism, which offers threat to the suffering patients.
Most of the AmpC producing isolates (n = 64) were recovered from patients suffering from wound infection (54.7%), urinary tract infection (20.3%), septicaemia (9.4%), ventilator assoicated (10.9%) and others (4.6%). In our study, although a proper methodological follow-up of the cases could not be done, but details derived from treatment sheets revealed that more than 50% patients suffering from urinary tract infection (UTI) and wound infection recovered and released from hospital. Mortality was reported in four cases from intensive care unit (ICU) and neonatal intensive care unit (NICU). However, the exact cause of death, whether due to any infections caused by resistant bacteria, could not be established.
To the best of our knowledge, this is the first report of clinical isolates of P. aeruginosa carrying blaPDC from India. The present study describes the resistance to expanded-spectrum cephalosporins in P. aeruginosa. Presence of additional genetic machinery showing expanded spectrum cephalosporin resistant phenotype would further complicate the antibiotic policy and therapeutic options in nosocomial infections caused by these isolates.
| ~ Acknowledgment|| |
The authors acknowledge the financial assistance provided by the Department of Biotechnology (Grant BT/PR11812/BRB/10/692), Government of India.
| ~ References|| |
|1.||Jacoby GA. AmpC beta-lactamases. Clin Microbiol Rev 2009;22:161-82. |
|2.||Livermore DM. Clinical significance of beta-lactamase induction and stable derepression in gram-negative rods. Eur J Clin Microbiol 1987;6:439-45. |
|3.||Livermore DM. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: Our worst nightmare? Clin Infect Dis 2002;34:634-40. |
|4.||Rodríguez-Martínez JM, Poirel L, Nordmann P. Extended-spectrum cephalosporinases in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2009;53:1766-71. |
|5.||Collee JG, Miles RS, Watt B. Tests for identification of bacteria. In: Collee JG, Marmion BP, Fraser AG, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. New York: Churchill Livingstone; 1996. p. 131-49. |
|6.||Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 18 th informational supplement. Clinical and Laboratory Standards, Wayne; 2008. |
|7.||Cantarelli VV, Inamine E, Brodt TC, Secchi C, Cavalcante BC, Pereira Fde S. Utility of the ceftazidime-imipenem antagonism test (CIAT) to detect and confirm the presence of inducible AmpC beta-Lactamases among enterobacteriaceae. Braz J Infect Dis 2007;11:237-9. |
|8.||Manchanda V, Singh NP. Occurrence and detection of AmpC beta-lactamases among Gram-negative clinical isolates using a modified three-dimensional test at Guru Tegh Bahadur Hospital, Delhi, India. J Antimicrob Chemother 2003;51:415-8. |
|9.||Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002;40:3798-801. |
|10.||Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002;40:2153-62. |
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]