|Year : 2016 | Volume
| Issue : 4 | Page : 433-441
Molecular characterisation of antimicrobial resistance in Pseudomonas aeruginosa and Acinetobacter baumannii during 2014 and 2015 collected across India
AK Pragasam1, S Vijayakumar1, YD Bakthavatchalam1, A Kapil2, BK Das2, P Ray3, V Gautam3, S Sistla4, SC Parija4, K Walia5, VC Ohri5, S Anandan1, B Veeraraghavan1
1 Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Microbiology, All Institute of Medical Sciences, New Delhi, India
3 Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
5 Division of Epidemiology and Communicable Diseases, n Council for Medical Research, New Delhi, India
|Date of Submission||13-Aug-2016|
|Date of Acceptance||14-Oct-2016|
|Date of Web Publication||8-Dec-2016|
Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Surveillance of antimicrobial resistance (AMR) is of great importance. Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens and emergence of resistance in these have increased the morbidity and mortality rates. This surveillance study was initiated by the Government of India - Indian Council of Medical Research. The aim of this study is to determine the antimicrobial susceptibility profile and to characterise the enzyme mediated antimicrobial resistance such as extended spectrum beta-lactamases (ESBLs) and carbapenemases among multidrug-resistant (MDR) P. aeruginosa and A. baumannii. Materials and Methods: A multi-centric study was conducted from January 2014 to December 2015 with a total number of 240 MDR P. aeruginosa and 312 MDR A. baumannii isolated from blood, cerebrospinal fluid, respiratory, pus, urine and intra-abdominal infections. Kirby–Bauer disc diffusion was done to determine the antimicrobial susceptibility profile. Further, MDR isolates were characterised by multiplex polymerase chain reaction to determine the resistance genes for ESBLs and carbapenemases. Results: Among the ESBLs, blaVEB (23%), blaTEM (5%) and blaSHV (0.4%) in P. aeruginosa and blaPER (54%), blaTEM (16%) and blaSHV (1%) in A. baumannii were the most prevalent. Likewise, blaVIM (37%), blaNDM (14%), blaGES (8%) and blaIMP (2%) in P. aeruginosa and blaOXA-23like (98%), blaOXA-58like (2%), blaNDM (22%) and blaVIM (3%) in A. baumannii were found to be the most prevalent carbapenemases. blaOXA-51like gene, intrinsic to A. baumannii was present in all the isolates tested. Conclusion: The data shown highlight the wide difference in the molecular mechanisms of AMR profile between P. aeruginosa and A. baumannii. In P. aeruginosa, plasmid-mediated mechanisms are much lesser than the chromosomal mediated mechanisms. In A. baumannii, class D oxacillinases are more common than other mechanisms. Continuous surveillance to monitor the trends in AMR among MDR pathogens is important for implementation of infection control and to guide appropriate empirical antimicrobial therapy.
Keywords: Surveillance, Antimicrobial resistance, β-lactamases, Pseudomonas aeruginosa, Acinetobacter baumannii
|How to cite this article:|
Pragasam A K, Vijayakumar S, Bakthavatchalam Y D, Kapil A, Das B K, Ray P, Gautam V, Sistla S, Parija S C, Walia K, Ohri V C, Anandan S, Veeraraghavan B. Molecular characterisation of antimicrobial resistance in Pseudomonas aeruginosa and Acinetobacter baumannii during 2014 and 2015 collected across India. Indian J Med Microbiol 2016;34:433-41
|How to cite this URL:|
Pragasam A K, Vijayakumar S, Bakthavatchalam Y D, Kapil A, Das B K, Ray P, Gautam V, Sistla S, Parija S C, Walia K, Ohri V C, Anandan S, Veeraraghavan B. Molecular characterisation of antimicrobial resistance in Pseudomonas aeruginosa and Acinetobacter baumannii during 2014 and 2015 collected across India. Indian J Med Microbiol [serial online] 2016 [cited 2017 Mar 28];34:433-41. Available from: http://www.ijmm.org/text.asp?2016/34/4/433/195376
| ~ Introduction|| |
Antimicrobial resistance is on the rise and it is a major public health problem across the world, and especially in developing countries like India. Infections caused by bacterial pathogens with multi, extremely and pan drug resistant phenotypes (MDR, XDR, PDR) are challenging and difficult to treat. This is due to the limited therapeutic options as very few agents such as carbapenems, colistin and tigecycline are available for treatment. However, treatment failure occurs and results in high morbidity and mortality rates with increase in health care costs. Studies on molecular characterisation of antimicrobial resistance mechanisms are limited in India. Surveillance studies across different geographical regions provide important information on trends in pathogen incidence and antimicrobial resistance. This information enables the targeted approaches in managing antimicrobial resistance.
Among the infections caused by Gram-negative bacteria, non-fermenting Gram-negative organisms such as Pseudomonas aeruginosa and Acinetobacter baumannii are troublesome. They cause wide range of infections such as bacteremia, pneumonia, skin and soft tissue infections and urinary tract infections. Infections caused by MDR, XDR and PDR P. aeruginosa and A. baumannii results in high morbidity and mortality rates, especially in immunocompromised individuals.
P. aeruginosa has the ability to resist wide range of anti-pseudomonal agents. It develops resistance through intrinsic and extrinsic resistance mechanisms. Intrinsic includes over-expression of efflux pumps (mexAB, mexCD, mexEF and mexXY), chromosomal hyper ampC producers and loss of porins (OprD); extrinsic includes acquisition of resistance genes such as extended spectrum beta-lactamases (ESBLs; blaSHV, blaTEM, blaVEB, blaPER and blaOXA types) and carbapenemases (blaGES, blaKPC, blaIMP, blaSPM, blaVIM and blaNDM)., Of these mechanisms, plasmid-mediated resistance genes are mostly studied due to its rapid dissemination. Conversely, chromosomal resistance has not been paid much attention. Mortality rate seen in P. aeruginosa pneumonia is up to 70%.
A. baumannii is resistant to almost all the available drugs and increased antimicrobial resistance has been implicated in nosocomial infections and hospital outbreaks. The mortality rates seen with A. baumannii caused pneumonia and blood stream infections were between 40%-70% and 28%–43%, respectively. Unlike P. aeruginosa, antimicrobial resistance in A. baumannii is predominantly through acquired resistance mechanisms such as production of ESBLs and class A carbapenemases (blaPER, blaTEM, blaSHV, blaGES and blaKPC), class B metallo β-lactamases (MBLs; blaNDM, blaVIM and blaIMP), class C β-lactamases (acinetobacter derived cephalosporinases) and the most common class D β-lactamases (blaOXA-23 like, blaOXA-24 like and blaOXA-58 like). Non-enzymatic mechanisms such as membrane impermeability by either loss of or decrease in expression of outer membrane proteins (CarO) or an increased expression of efflux pumps (AdeABC) also contributes to antimicrobial resistance in A. baumannii.,, Studies have reported that co-occurrence of class D β-lactamases and overexpression of efflux system contribute to increased resistance in A. baumannii.
As there is a great need for surveillance studies in India, the Indian Council of Medical Research (ICMR) undertook this surveillance study across four different centres from India. This includes All India Institute of Medical Sciences (AIIMS), Delhi; Christian Medical College (CMC), Vellore; Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry (JIPMER) and Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh. The main purpose of this study was to (i) establish “Antimicrobial Resistance Surveillance Network” in India; (ii) to monitor the antimicrobial susceptibility profile of clinical isolates; and (iii) to look for the prevalence of various enzymes conferring resistance in clinical isolates. Herein we report the molecular resistance mechanisms of clinical isolates of P. aeruginosa and A. baumannii collected across India during 2014–2015.
| ~ Materials and Methods|| |
A total of 552 clinically significant, non-duplicate isolates of P. aeruginosa (n = 240) and A. baumannii (n = 312) were received from AIIMS (New Delhi), CMC (Vellore), JIPMER (Puducherry) and PGIMER (Chandigarh) during January 2014–December 2015. Among 240 P. aeruginosa isolates, 125, 55, 36, 13, 7 and 3 were from respiratory secretions (broncho-alveolar lavage and endotracheal secretions), blood, pus, intra-abdominal secretions, urine and cerebrospinal fluid specimens, respectively. Similarly, among 312 A. baumannii isolates, 165, 77, 37, 22, 6 and 5 were from respiratory secretions, blood, pus, intra-abdominal secretions, CSF and urine specimens, respectively. The organisms were identified up to the species level using standard biochemical tests. Further confirmation of A. baumannii was done using blaOXA-51Like polymerase chain reaction (PCR) which is intrinsic to this species.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing for different classes of antimicrobials such as cephalosporins (cefotaxime, ceftazidime); β-lactam/β-lactamase inhibitors (piperacillin/tazobactam); carbapenems (imipenem, meropenem); fluoroquinolones (ciprofloxacin, levofloxacin) and PB300 units; tigecycline (only for A. baumannii) was performed for all the isolates by Kirby Bauer disk diffusion method and interpreted according to Clinical Laboratory Standards Institute guidelines 2014 and 2015., Isolates showing resistance to at least one agent in each of these three or four groups (cephalosporins, carbapenems, fluoroquinolones and/or aminoglycosides) were considered as MDR and included in the study for further characterisation.
Carba NP (for P. aeruginosa): A subset of 111 isolates of carbapenem-resistant P. aeruginosa was randomly chosen and subjected to Carba NP test as described previously. The isolates were grown on Mueller-Hinton agar plates for 24 h and isolated colonies were subjected for Carba NP testing.
CarbAcineto NP test (for A. baumannii):
A subset of 105 isolates of carbapenem resistant A. baumannii was randomly chosen, and a modified protocol of Carba NP known as CarbAcineto NP test was used. Class D oxacillinases are more prevalent in Acinetobacter spp., a reformed protocol using 5 M NaCl was used instead of BPER-II lysis buffer, to avoid any buffer effect, as blaOXA enzymes possess weak carbapenemase activity.
For both Carba NP and CarbAcineto NP tests, Klebsiella pneumoniae ATCC BAA 1705 and Klebsiella pneumoniae ATCC BAA 1706 were used as positive and negative controls, respectively, in all the assays. Change of the colour of phenol red indicator from red to yellow was taken as positive for carbapenemase production. The absence of colour change from red to yellow was taken as negative result. The observation of colour change was taken by two independent readers.
Imipenem + cloxacillin test (only for P. aeruginosa): Randomly chosen 53 isolates of P. aeruginosa were tested for imipenem and cloxacillin synergism testing. The most common non-carbapenemase mediated resistance mechanism in P. aeruginosa is porin loss with hyperproduction of chromosomal ampC enzymes. Combination disc test was done for imipenem and cloxacillin as described previously, to demonstrate the ampC production. Cloxacillin of 4000 µg/ml was used alone and in combination with imipenem (10 µg/ml). More than 5 mm difference in the zone diameter between imipenem alone and in combination of imipenem with cloxacillin was taken as positive result for chromosomal hyper ampC producers in P. aeruginosa.
Multiplex polymerase chain reaction for detection of extended spectrum beta-lactamases and carbapenemase genes
All the test isolates were grown on blood agar overnight, and genomic DNA was extracted by boiling lysis method. Conventional multiplex PCR was done for the detection of ESBL genes such as blaTEM, blaSHV, blaPER, and blaVEB, and carbapenemase genes such as blaGES, blaSPM, blaIMP, blaVIM, blaNDM, blaKPC, blaOXA-48 like as described previously; and blaSIMLike (Only for A. baumannii). In addition, the presence of class D carbapenemase genes such as blaOXA-23 like, blaOXA-24 like and blaOXA-58 like were screened for A. baumannii isolates by multiplex PCR. The amplicons were visualised in 2% agarose gel with staining by ethidium bromide. Known positive controls for appropriate genes were used for every run (courtesy: IHMA, Inc., USA).
| ~ Results|| |
Phenotypic test results – CarbaNP
A total of 111 isolates were screened for carbapenemase production by Carba NP test. 37% (n = 41) of isolates showed Carba NP test positive and 63% (n = 70) of isolates showed Carba NP test negative. Among the Carba NP positives (n = 41), 81% were found to be due to the carbapenemase production such as blaNDM, blaVIM and blaVIM + NDM, while 19% were negative in PCR for the carbapenemase genes tested. Similarly, for Carba NP negatives (n = 70), carbapenemase genes were found in 17% (n = 12) of isolates producing blaGES, blaNDM, blaVIM and blaVIM+NDM; while 83% (n = 58) were negatives in PCR for the carbapenemase genes tested.
AmpC hyperproduction analysis was carried out for a subset of randomly chosen isolates (n = 53). Among the 53 isolates tested, 36% (n = 19) were carbapenemase producers; and 64% (n = 34) were non-carbapenemase producers. Notably, 26% (n = 5/19) of the carbapenemase producers showed positive for ampC hyperproduction (this includes 45% (n = 5/11) of blaVIM producers). 74% were negative, which includes blaVIM (n = 6) and NDM (n = 8). Among the non-carbapenemase producers, 82% (n = 28/34) were found to be positive for ampC hyper production tests, while 18% (n = 6/34) were negative.
Genotypic results of β lactamases
Extended spectrum beta lactamases
Among the 240 P. aeruginosa screened, 29% (n = 69) of the isolates were positive for the ESBL genes tested. This includes, 23% (n = 56) of blaVEB, 5% (n = 12) of blaTEM and 0.4% (n = 1) of blaSHV. Of the ESBL genes, blaVEB was found to be the most common followed by blaTEM and blaSHV. Co-producers of blaSHV and blaTEM were also found. However, among the ESBL positives, geographical distribution was found to be almost similar for blaTEM. Notably, all the isolates (n = 21) from PGIMER were positive for blaTEM gene. blaSHV was identified only in CMC isoaltes, while other ESBLs have been identified across all the four study centres. Overall, ESBL rates were ranged from 15% to 100% across the study sites, with 23%, 24%, 15% and 100% in AIIMS, CMC, JIPMER and PGIMER, respectively. This varying ESBL rates could be due to the unequal number of isolates tested from each of the study centres.
Among the 240 P. aeruginosa screened, 8% (n = 18) were of class A (blaGES) carbapenemase and 53% (n = 127) class B carbapenemases (MBLs - blaVIM, blaNDM and blaIMP). Class D carbapenemases (blaOXA-48 like) was not detected in any of the isolates tested. Geographical distribution of carbapenemase classes varied among the study centres. Class A carbapenemases were not detected in AIIMS, while 6%, 12% and 10% were detected in CMC, JIPMER and PGIMER, respectively. However, among the class B carbapenemases (MBLs), blaIMP, blaVIM, blaNDM was identified except blaSPM. Within that, blaVIM were detected in 24%–57%, blaNDM were 8%–19% and blaIMP were 4%–5%, respectively. blaNDM was not observed among PGIMER isolates. Overall, the carbapenemase rates were ranged from 6% to 12% and 38%–75% for class A and class B carbapenemases, respectively. Interestingly, from JIPMER study isolates, 75% were MBL producers, this is alarming. While 54%, 38% and 57% MBLs were detected in AIIMS, CMC and PGIMER isolates, respectively. These differences in the rates of carbapenemases could be due to the unequal number of isolates tested from each centres. Molecular profiles of β-lactamases in P. aeruginosa are summarised in [Table 1].
|Table 1: Extended spectrum β lactamases and carbapenemases seen in clinical isolates of Pseudomonas aeruginosa in India|
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Phenotypic test results-CarbAcineto NP
CarbAcineto NP test was done for a total of 105 isolates of A. baumannii. Of which, 88% (n = 92) of the isolates were positive and 12% (n = 13) were negative. Among the 92 positive isolates, 91 isolates were positive for class D blaOXA-23 like gene and 13 isolates were positive for one of the following class B carbapenemases, blaNDM and/or blaVIM gene. Of the 13 CarbAcineto NP negatives, blaOXA-23 like gene was present in all the isolates, whereas one isolate was positive for either blaNDM or blaVIM gene.
Genotypic results of β lactamases
Extended spectrum beta lactamases
Among the 312 A. baumannii screened, 71% (n = 223) of the isolates were positive for the ESBL genes tested. This includes, 54% (n = 169) of blaPER, 16% (n = 50) of blaTEM and 1% (n = 4) of blaSHV. blaPER was found to be the most common ESBL gene followed by blaTEM and blaSHV. All the study isolates were negative for blaVEB. In addition, co-producers of blaPER, blaTEM, and blaNDM, were found. Geographical distribution of both blaPER and blaTEM was found to be almost similar across the four study centres, whereas blaSHV was identified in CMC and PGIMER isolates.
Class A carbapenemases (blaGES and blaKPC) was not detected in any of the study isolates tested. Twenty-five percent (n = 78) of the isolates were positive for class B carbapenemases, of which 22% were blaNDM (n = 69) followed by 3% of blaVIM (n = 9). None of the isolates harboured blaIMP, blaSPM and blaSIM genes. Intrinsic class D carbapenemase, blaOXA-51 like was found in all the study isolates whereas acquired class D carbapenemases such as blaOXA-23 like and blaOXA-58 like was found to be 98% (n = 306) and 2% (n = 6), respectively. blaOXA-48 like and blaOXA-24 like was not detected in any of the isolates tested. Geographical distribution was found to be almost similar for both blaOXA-51 like and blaOXA-23 like across all the four study centres, whereas blaOXA-58 like was found in AIIMS, CMC and JIPMER isolates. Molecular profiles of β lactamases in A. baumannii are summarised in [Table 2].
|Table 2: Extended spectrum beta-lactamases and carbapenemases seen in clinical isolates of Acinetobacter baumannii in India|
Click here to view
| ~ Discussion|| |
Antimicrobial resistance surveillance plays an important role in tracking resistance to various antimicrobial agents. Active surveillance has been carried out by various studies including ANSORP, SMART, ABCS, SCOPE, SENTRY, MYSTIC, TEST, CHINET and EARS. Of all these studies, India being a participating centre is very less [Table 3]. [Table 4] and [Table 5] summarise the cumulative report of molecular resistance mechanisms carried out in various Indian hospitals. To address this gap, ICMR has initiated the network to document and monitor the antimicrobial resistance across various regions in India.
|Table 3: Consolidated global surveillance study reports on molecular resistance mechanisms seen among Acinetobacter baumannii and Pseudomonas aeruginosa|
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|Table 4: Molecular characterisation of enzyme-mediated resistance mechanisms in Pseudomonas aeruginosa reported in Indian studies|
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|Table 5: Molecular characterisation of enzyme mediated resistance mechanisms in Acinetobacter baumannii reported in Indian studies|
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MDR rates in P. aeruginosa ranged from 20% to 30% and in A. baumannii ranged from 40% to 50% in India (unpublished data). In P. aeruginosa, molecular analysis of ESBL revealed the presence of blaVEB, blaTEM and blaSHV. Among the ESBLs, blaVEB was the most common followed by blaTEM and blaSHV. Notably, regional distribution of ESBLs varied across regions. In particular, PGIMER isolates showed 100% (n = 21/21) blaVEB positivity, while in other centres it ranged from 12% to 19%. Molecular profile of ESBL genes by other Indian studies shows similar profile for blaTEM and blaSHV genes., However, blaPER gene was not seen in this study isolates of P. aeruginosa, which is in contrast to 17% reported.
Carbapenem resistance in P. aeruginosa due to carbapenemase was found to be 44%. Of the carbapenemases, blaVIM is the most common MBL identified. This observation is in concurrence with the previous reports, wherein, blaVIM mediated resistance is common in P. aeruginosa.,,,,,,, Although blaNDM is widespread across India, the isolation rate of blaNDM in P. aeruginosa is less. This study detected about 8%–19%, while PGIMER isolates did not harbour blaNDM. These regional variation needs to be further confirmed with testing of more number of isolates. However, this finding is similar with the other Indian studies, wherein the rates of blaNDM ranged from 5% to 24%.,, In additional, ampC mediated resistance to carbapenems are reported at much higher rates than carbapenemases. In this study, subset analysis of non-carbapenemase producers showed that 82% of carbapenem resistance was contributed by porin loss and/or chromosomal ampC hyper production, which is also confirmed by Upadhyay et al., of about 43%.
In A. baumannii, blaPER (54%) was the most common ESBL followed by blaTEM and blaSHV. The incidence of blaPER was found to be similar across all the study centres. Carbapenem resistance in A. baumannii is most predominantly occurs due to chromosomal/plasmid-mediated class D oxacillinases (blaOXA-51 like and blaOXA-23 like) followed by class B MBL (blaNDM and blaVIM), respectively. In this study, carbapenem resistance due to blaOXA-23like was found to be 98%, which shows concordance with other studies ,,,, whereas blaNDM and blaVIM were about 22% and 3%, respectively.,
| ~ Conclusion|| |
From this study, it was clear that ESBLs and carbapenemases detected from the clinical isolates varies widely for P. aeruginosa and A. baumannii. In P. aeruginosa, plasmid-mediated mechanisms are much lesser than the chromosomal mediated mechanisms. In A. baumannii, class D oxacillinases are more common, than the other mechanisms. Continuous surveillance will help to monitor and control the spread of antimicrobial resistance. Also, the cumulative national data will help policy makers to advice on appropriate empirical treatment guideline. Further, this report highlights the β-lactamase profile in P. aeruginosa and A. baumannii clinical isolates seen in India. Based on this, the suitable β-lactamase inhibitor can be recommended in combination with β-lactams. However, these inhibitors based combinations needs to be evaluated and monitored. This includes avibactam, which is clinically active against Class A and C β-lactamases seen in P. aeruginosa and aztreonam with avibactam which is active against MBL producers. Whereas for class D OXA carbapenemases (blaOXA-23/24/51/58 like), it needs to be explored.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]