|Year : 2018 | Volume
| Issue : 3 | Page : 344-351
Dominance of international high-risk clones in carbapenemase-producing Pseudomonas aeruginosa: Multicentric molecular epidemiology report from India
Agila Kumari Pragasam1, Balaji Veeraraghavan1, Shalini Anandan1, Vignesh Narasiman1, Sujatha Sistla2, Arti Kapil3, Purva Mathur3, Pallab Ray4, Chand Wattal5, Sanjay Bhattacharya6, Vijayashri Deotale7, K Subramani8, JV Peter8, TD Hariharan9, I Ramya10, S Iniyan11, Kamini Walia12, VC Ohri12
1 Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
3 Department of Microbiology, All India Institute of Medical Science, New Delhi, India
4 Department of Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
5 Department of Microbiology, Sir Ganga Ram Hospital, New Delhi, India
6 Department of Microbiology, Tata Medical Centre, Kolkatta, West Bengal, India
7 Department of Microbiology, Mahatma Gandhi Institute of Medical Science, Sevagram, Maharashtra, India
8 Department of Critical Care, Christian Medical College, Vellore, Tamil Nadu, India
9 Department of Orthopaedic Surgery, Christian Medical College, Vellore, Tamil Nadu, India
10 Department of Medicine (Unit-5), Christian Medical College, Vellore, Tamil Nadu, India
11 Department of Surgery, Christian Medical College, Vellore, Tamil Nadu, India
12 Division of Epidemiology and Communicable Disease, Department of Microbiology, Indian Council of Medical Research, New Delhi, India
|Date of Web Publication||14-Nov-2018|
Dr. Balaji Veeraraghavan
Department of Clinical Microbiology, Christian Medical College, Vellore - 632 004, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Pseudomonas aeruginosa is one of the most common opportunistic pathogens that cause severe infections in humans. The burden of carbapenem resistance is particularly high and is on the rise. Very little information is available on the molecular mechanisms and its clonal types of carbapenem-resistant P. aeruginosa seen in Indian hospitals. This study was undertaken to monitor the β-lactamase profile and to investigate the genetic relatedness of the carbapenemase-producing (CP) P. aeruginosa collected across different hospitals from India. Materials and Methods: A total of 507 non-duplicate, carbapenem-resistant P. aeruginosa isolated from various clinical specimens collected during 2014–2017 across seven Indian hospitals were included. Conventional multiplex polymerase chain reaction for the genes encoding beta-lactamases such as extended-spectrum beta-lactamase (ESBL) and carbapenemase were screened. A subset of isolates (n = 133) of CP P. aeruginosa were genotyped by multilocus sequence typing (MLST) scheme. Results: Of the total 507 isolates, 15%, 40% and 20% were positive for genes encoding ESBLs, carbapenemases and ESBLs + carbapenemases, respectively, whilst 25% were negative for the β-lactamases screened. Amongst the ESBL genes, blaVEB is the most predominant, followed by blaPER and blaTEM, whilst blaVIM and blaNDM were the most predominant carbapenemases seen. However, regional differences were noted in the β-lactamases profile across the study sites. Genotyping by MLST revealed 54 different sequence types (STs). The most common are ST357, ST235, ST233 and ST244. Six clonal complexes were found (CC357, CC235, CC244, CC1047, CC664 and CC308). About 24% of total STs are of novel types and these were found to emerge from the high-risk clones. Conclusion: This is the first large study from India to report the baseline data on the molecular resistance mechanisms and its association with genetic relatedness of CP P. aeruginosa circulating in Indian hospitals. blaVIM- and blaNDM-producing P. aeruginosa is the most prevalent carbapenemase seen in India. Majority of the isolates belongs to the high-risk international clones ST235, ST357 and ST664 which is a concern.
Keywords: Carbapenem, clonality, high-risk clones, Pseudomonas aeruginosa, resistance
|How to cite this article:|
Pragasam AK, Veeraraghavan B, Anandan S, Narasiman V, Sistla S, Kapil A, Mathur P, Ray P, Wattal C, Bhattacharya S, Deotale V, Subramani K, Peter J V, Hariharan T D, Ramya I, Iniyan S, Walia K, Ohri V C. Dominance of international high-risk clones in carbapenemase-producing Pseudomonas aeruginosa: Multicentric molecular epidemiology report from India. Indian J Med Microbiol 2018;36:344-51
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Pragasam AK, Veeraraghavan B, Anandan S, Narasiman V, Sistla S, Kapil A, Mathur P, Ray P, Wattal C, Bhattacharya S, Deotale V, Subramani K, Peter J V, Hariharan T D, Ramya I, Iniyan S, Walia K, Ohri V C. Dominance of international high-risk clones in carbapenemase-producing Pseudomonas aeruginosa: Multicentric molecular epidemiology report from India. Indian J Med Microbiol [serial online] 2018 [cited 2019 Aug 23];36:344-51. Available from: http://www.ijmm.org/text.asp?2018/36/3/344/245388
| ~ Introduction|| |
Pseudomonas aeruginosa is one of the most common opportunistic pathogens that cause severe infections in humans. The burden rate is especially high in patients admitted to intensive care units, burn centres and patients with cystic fibrosis. High rates of multidrug-resistant P. aeruginosa infections is challenging for treatment. This is due to the ability of this pathogen to resist to a wide range of anti-pseudomonal agents through multiple defence mechanisms.,
Antimicrobial resistance (AMR) mechanisms is by both intrinsic (chromosomal) and acquired (AMR determinants through plasmids). Chromosomal is due to the production of AmpC enzyme, alteration/inactivation of OprD and overexpression of efflux pumps which confers resistance to anti-pseudomonal β-lactam agents (ceftazidime, imipenem and meropenem), fluoroquinolones (levofloxacin) and aminoglycosides (amikacin and gentamicin). In addition, acquisition of plasmid-mediated extended-spectrum beta-lactamases (ESBLs-blaVEB, blaTEM) and carbapenemase (blaVIM, blaNDM, blaGIM, blaSIM, blaGES and blaIMP) are also frequently reported in P. aeruginosa. These β-lactamases are reported to be carried on the integrons and hence the widespread dissemination.
Carbapenems are important agents used for the management of infections caused by drug-resistant P. aeruginosa. However, increasing resistance to these agent limits its use. There is a little information available on the molecular mechanisms of carbapenem resistance in P. aeruginosa seen in Indian hospitals. Studies have reported blaVIM and blaNDM to be the predominant AMR determinants contributing for carbapenem resistance.
In addition to the molecular mechanisms, understanding the genetic relatedness with varying carbapenemase profile would provide insights into the clonality of this pathogen. Earlier studies have shown P. aeruginosa is typically non-clonal. Whilst, metallo-β-lactamase-producing isolates in a hospital setting is reported to be clonal, indicating the hospital-acquired infections. These includes the well known “international high-risk clones” of multidrug-resistant/extensively drug-resistant (MDR/XDR) P. aeruginosa belonging to ST235, ST111 and ST175.
Given the increasing prevalence of MDR/XDR carbapenemase-producing (CP) P. aeruginosa, no data is available till date from India on the molecular mechanism and its genetic relatedness. As a part of the Indian Council of Medical Research (ICMR)-AMR Surveillance Network (AMRSN), this study was undertaken to monitor the β-lactamase profile and to investigate the epidemiology of the CP P. aeruginosa collected across different hospitals from India.
| ~ Materials and Methods|| |
A total of 507 non-duplicate, carbapenem-resistant P. aeruginosa isolated from various clinical specimens collected during 2014–2017 were included. These were collected from the hospitals that were the part of ICMR-AMRSN. It includes All India Institute of Medical Sciences, Delhi; Christian Medical College, Vellore (CMC); Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry (JIPMER); Mahatma Gandhi Institute of Medical Sciences, Maharashtra; Postgraduate Institute of Medical Education and Research, Chandigarh (PGIMER); Sir Gangaram Hospital, Delhi; Tata Medical Centre, Kolkata. Amongst 507 P. aeruginosa isolates, 125, 55, 36, 13, 7 and 3 were from respiratory secretions (bronchoalveolar lavage and endotracheal secretions), blood, pus, intra-abdominal secretions, urine and cerebrospinal fluid specimens, respectively. The organisms were identified up to the species level using standard biochemical tests.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing against the anti-pseudomonal agents was tested. This includes cephalosporins (ceftazidime-30 μg, cefepime-30 μg); β-lactam/β-lactamase inhibitors (piperacillin/tazobactam-100/10 μg, cefoperazone/sulbactam-30/10 μg); carbapenems (imipenem-10 μg, meropenem-10 μg); fluoroquinolones (ciprofloxacin-5 μg, levofloxacin-5 μg) and aminoglycosides (amikacin-30 μg/gentamicin-10 μg/tobramycin-10 μg). Susceptibility was determined by disc diffusion for all the isolates by Kirby–Bauer disc diffusion method according to CLSI (Clinical Laboratory Standards Institute) guidelines 2014–2017 (M100-S24-27). Isolates resistant to carbapenems (imipenem and meropenem) were included in the study for further characterisation. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality controls and their zone of inhibition was within recommended range.
Multiplex polymerase chain reaction for detection of extended-spectrum beta-lactamase and carbapenemase genes
All the test isolates were grown on blood agar overnight and whole genomic DNA was extracted by boiling lysis method. Conventional multiplex polymerase chain reaction was done for the acquired carbapenemase encoding genes such as blaGES, blaSPM, blaIMP, blaVIM, blaNDM, blaKPC and blaOXA-48like as described previously. Known positive controls for each gene were used in every run (control strains courtesy-IHMA Inc.,).
Multilocus sequence typing
A total of 156 isolate was characterized for their genotypes by multilocus sequence typing (MLST) as described by pubMLST scheme (http://pubmlst. org/paeruginosa). DNA amplification of the seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA and trpE) was performed for all isolates. Amplicons were sequenced in both directions using the DNA analyser ABI (Applied Biosystems, Foster City, CA). The nucleotide sequences of the study isolates were compared to existing sequences deposited in the MLST database to identify their allelic numbers, so as the find the sequence types (STs). eBURST v3.0 was used to perform the phylogenetic analysis of the studied population in comparison to the 3053 STs available in the database (accessed on April 22, 2018). Novel alleles and novel STs were submitted to the MLST database and to assign an allele number and an STs by the database curator.
| ~ Results|| |
Of the total 507 isolates, 15%, 40% and 20% were positive for genes encoding ESBLs, carbapenemases and ESBLs + carbapenemases, respectively, whilst 25% were negative for the β-lactamases screened. Amongst the ESBL genes, blaVEB is the most predominant, followed by blaPER and blaTEM. blaPER is seen only in JIPMER isolates collected during 2016, indicating the regional variation. Amongst the carbapenemase, 33% had single carbapenemases, such as blaVIM (21%), followed by blaNDM (8%), blaGES (3%) and blaIMP (1%). Whilst 6.4% had double carbapenemases, this includes blaNDM + VIM (3%), blaNDM + GES (2%), blaVIM + GES (1%), blaIMP + VIM (0.2%), blaIMP + NDM (0.2%) and 1% of triple carbapenemases genes, blaIMP + VIM + GES (0.6%) and blaIMP + VIM + NDM (0.2%). Among the co-producers of ESBLs and Carbapenemases, 13% of isolates had combinations of one gene each of ESBL and carbapenemase. Whereas, 5.6% had multiple ESBL genes with carbapenemases. Carbapenemase profile seen across the study sites are mentioned in [Figure 1] and [Table 1].
|Figure 1: Carbapenemase prevalence rates in the antimicrobial resistance surveillance network sites in India. Star marks indicate the participating sites in the network. Overall carbapenemase rates with individual carbapenemase rates are given for each of the centres|
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|Table 1: Carbapenemase profiles in Pseudomonas aeruginosa collection across Indian hospitals|
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Epidemiology of carbapenemase-producing Pseudomonas aeruginosa
Amongst the 156 CP P. aeruginosa, a total of 61 different ST's were observed. 50% (n = 77) of the total ST's belong to the five most commonly observed ST's associated with drug resistance, such as ST357 (n = 32, 21%), ST235 (n = 15, 10%), ST244 (n = 15, 10%), ST233 (n = 10, 7%) and ST644 (n = 5, 3%). Whilst, 30% (n = 40) of the ST's were polyclonal and highly diverse. The study isolates formed six clusters as clonal complexes (CC's). This includes CC235 (ST534, ST1081, ST2645, ST2577, ST2637, ST3022), CC357 (ST2579, ST3026, ST1831, ST1618, ST3025, ST3077), CC244 (ST2266, ST2609), CC1047 (ST2608, ST2573, ST2654), CC664 (ST2606, ST2610, ST2736) and CC308 (ST481, ST3021). The CC's were depicted in [Figure 2].
|Figure 2: Network of 3076 sequence types listed in the PubMLST database (as on August 7, 2018). Each black dot indicates the sequence type, lines connecting the dots indicates the single-loci variant's or double-loci variant's emerged from the founder sequence which is with blue dot. Sequence type number in magenta is the founder of the clonal complex and sequence type numbers in green are found in this study isolates|
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Emerging novel types in the studied population
Notably, 24% (n = 38) of the ST's were of novel types, amongst which 21, 9 and 3 were single-loci variant (SLV), double-loci variants (DLVs) and triple-loci variants of the existing predominant ST's, respectively. Notably, >80% of the SLV's have emerged from the well-known high-risk clones such as ST235, ST357, ST644 and ST1047. Whilst, ST's emerged from DLV's were of different STs. Regional variations in the ST's and the detailed profile of the novel STs are summarised in [Table 2]. Interestingly, population structure from CMC, TATA Medical Centre and PGIMER were similar to high-risk clones. However, 55% (n = 21/38) of the total novel ST's were from JIPMER isolates, which makes the population highly diverse.
|Table 2: Profile of carbapenemase-producing Pseudomonas aeruginosa with new/novel sequence type's reported in this study|
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Association of carbapenemase with the sequence types
Although the population structure is polyclonal and diverse, some degree of similarity is observed amongst the high-risk clones that carried the carbapenemase. Notably, amongst the blaVIM producers, six different clones have been observed (ST233, ST244, ST664, ST773, ST823 and ST357). Notably, isolates of ST235 had more than one carbapenemases, along with other ST's such as ST1047 and ST308 and their SLV's and DLV's, respectively. Whilst in blaNDM, ST357 was the predominant, followed by ST308 and ST1203. Each ST's harboured a variety of carbapenemase, signifying the ability of these prevailing clones to acquire the AMR determinants. Across all the study sites, isolates from CMC were comparatively clonal than other sites, where ST357, ST244 and ST233 harboured blaNDM and/orblaVIM but not any other carbapenemases. This substantiates the replacement of mixed population by these prevailing high-risk clones, which is alarming. On the other hand, isolates producing blaIMP were present only in isolates from JIPMER and SIR Gangaram. Whilst, very few Class A carbapenemase were detected, which was predominantly confined to ST1203 and its SLV's and DLV's. Details on the MLST profile are shown in the phylogenetic tree in the [Figure 3].
|Figure 3: Unrooted tree of concatenated sequence of the 7 housekeeping gene sequences of multilocus sequence typing scheme obtained using MEGA 7.0 and labelled with the metadata using iTOL.embl. Scale bar 0.01 indicates the number of nucleotide substitution per site, which is the amount of genetic change within the tested population|
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| ~ Discussion|| |
Globally, blaVIM is the most commonly encountered carbapenemase in P. aeruginosa. In addition, other carbapenemases such as blaKPC, blaGES, blaIMP, blaNDMblaSPM have also been reported. However, blaVIM-mediated carbapenem resistance was previously reported from Indian isolates., This further concurs with the other study isolates of India and its neighbouring countries (SEARO region).
This is the first large study from India to report the carbapenemase profile and its genetic relatedness in P. aeruginosa at a national level. It provides further evidence to the existing knowledge on the epidemiology of the CP-P. aeruginosa, which is dynamic. Although previous studies report the population structure of P. aeruginosa to be highly diverse, we report the emerging clones with novel ST's. These are SLV's or DLV's of the prevailing international high-risk clones, which is a significant observation. This process of emerging new clones from the prevailing clones is a serious threat as they carry drug-resistant determinants. This emergence and evolution in this deadly pathogen needs to be constantly monitored for the occurrence of susceptible clones being replaced. Whole genome-based SNP typing would provide insights into the variations across the entire genome within the ST's, which favours the evolution. Such studies warrant the better understanding of the evolution of this pathogen which undergoes dynamic changes making their population diverse. This would help in implementing strict infection control strategies to prevent and control the spread of these high-risk clones to be endemic in Indian hospitals.
As there were abundant literatures on non-clonal population structure of P. aeruginosa, we undertook this study to specifically investigate the clonal distribution of CP P. aeruginosa isolated in the hospital settings across India. Amongst the various clones encountered in this study, ST357, ST235 and ST111 are the concerning clones, which is of high risk. ST235 is the most prevalent and widespread international clone across the world, which has emerged in Europe around 1980's.,, Remarkably, a recent study on the whole genome-based analysis of ST235 genomes have provided much more insights in terms of its clinical significance. A set of 22 ST-235-specific genes signatures were identified, which contributes for its virulence (especially exoU-encoded endotoxin) and ability for acquisition of AMR determinants through mobile genetic elements. This implicated in the severity of the diseases and poor outcome.
Another significant finding is the population structure, which was found to be clonal amongst the CP P. aeruginosa. Although 45 different STs were identified, about 65% of the isolates was found to be clonal with four major CC's, whilst 40% were diverse and non-clonal. The diverse clonality observed was due to the emergence of novel ST's from the high risk clones, which is due to the SNPs. This must be evaluated to understand the population structure to be clonal or diverse. Such finding concurs with the clonality profile amongst MDR/XDR P. aeruginosa, whilst diverse amongst the antibiotic susceptible phenotypes., However, MLST-based SNP could provide only limited information on the population expansion, whilst whole genome SNP could provide further insights on the evolution of this pathogen. Future studies must focus on analysing the whole genome-based typing to understand the genetic basis for evolution.
One major limitation of the study is that, carbapenem-resistant (CR) P. aeruginosa with resistance profiles such loss of porin/overexpression of efflux pumps have not been investigated, as these are the second most commonly seen phenotypes. Knowledge on population structure of such phenotypes is lacking, which is also important. Further, for the non-carbapenemase-mediated CR P. aeruginosa, studies differentiating the clonality on a whole-genome level are essential. This may help in understanding the genomic signatures influencing the acquisition of AMR determinants and/or development of chromosomal-mediated resistance amongst CR P. aeruginosa.
The present study reports baseline data on the molecular resistance mechanisms and its association with genetic relatedness of CP P. aeruginosa circulating in Indian hospitals. blaVIMand blaNDM-producing P. aeruginosa is the most prevalent carbapenemase seen in India. Majority of the isolates belongs to the high-risk international clones ST235, ST357, ST244 and ST235 which concurs with the earlier reports. Continuous monitoring is necessary to better understand the clonal distribution with the replacement phenomenon of susceptible clones with high-risk clones and to implement appropriate prevention strategies.
Financial support and sponsorship
This study was financially supported by the Indian Council of Medical Research (ICMR - Ref: file no: AMR/TF/54/13ECDH11).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]