|Year : 2018 | Volume
| Issue : 4 | Page : 250-255
Prevalence of New Delhi metallo-β-lactamase-1 in Pseudomonas aeruginosa and its antimicrobial resistance profile in hospitalized patients in a tertiary care hospital setup
Anjali Agarwal1, Sneha Mohan2, Ujjwal Maheshwari3, Sambhav Jain4, Suresh Lacchhman Akulwar5
1 Department of Microbiology, Hind Institute of Medical Sciences, Barabanki, Uttar Pradesh, India
2 Department of Microbiology, School of Medical Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
3 Department of Cardiology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
4 Private Practice, Sangrur, Punjab, India
5 Department of Microbiology, Hind Institute of Medical Sciences, Mau, Ataria, Sitapur, Uttar Pradesh, India
|Date of Web Publication||15-Oct-2018|
Dr. Anjali Agarwal
Department of Microbiology, Hind Institute of Medical Sciences, Barabanki, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
BACKGROUND: Pseudomonas aeruginosa is one of the most common causes of nosocomial infections. Carbapenems are widely used for the treatment of this microorganism, but nowadays, increasing number of carbapenem-resistant P. aeruginosa (CRPA) isolates are increasing which further lead to treatment failure due to associated multidrug resistance.
AIM: The present study was done to determine the prevalence of New Delhi metallo-β-lactamase-1 (bla NDM-1) in P. aeruginosa in hospitalized patients in a tertiary care setup.
SETTINGS AND DESIGN: The study was conducted in the department of microbiology with the tertiary care hospital settings. It was a prospective cross-sectional observational study conducted from June 2014 to December 2014.
MATERIALS AND METHODS: Antibiotic susceptibility test was performed using Kirby–Bauer disc diffusion method for screening of the isolates. P. aeruginosa isolates with the zone diameter of carbapenems ≤18 mm were considered as carbapenemase producers. Metallo-beta-lactamases (MBL) detection was done using imipenem- ethylenediaminetetraacetic acid double-disc synergy test. NDM-1 gene in P. aeruginosa was identified using conventional polymerase chain reaction.
STATISTICAL ANALYSIS USED: Chi-square test was used to analyze the data and P < 0.01 was considered statistically significant.
RESULTS: Out of 460 P. aeruginosa, 74 (16.08%) isolates were CRPA. A total of 61 (13.2%) MBL-producing P. aeruginosa were isolated, and bla NDM-1 gene was detected in 18 (3.91%) P. aeruginosa isolates.
CONCLUSION: The study showed increasing multidrug resistance among CRPA compared to carbapenem-sensitive P. aeruginosa. It also determined increasing prevalence of bla NDM-1 in P. aeruginosa isolates. It is emphasized to timely identify these pathogens and treat with proper antibiotics to reduce morbidity and mortality.
Keywords: Carbapenemase, New Delhi metallo-β-lactamase-1, Pseudomonas aeruginosa
|How to cite this article:|
Agarwal A, Mohan S, Maheshwari U, Jain S, Akulwar SL. Prevalence of New Delhi metallo-β-lactamase-1 in Pseudomonas aeruginosa and its antimicrobial resistance profile in hospitalized patients in a tertiary care hospital setup. Int J Health Allied Sci 2018;7:250-5
|How to cite this URL:|
Agarwal A, Mohan S, Maheshwari U, Jain S, Akulwar SL. Prevalence of New Delhi metallo-β-lactamase-1 in Pseudomonas aeruginosa and its antimicrobial resistance profile in hospitalized patients in a tertiary care hospital setup. Int J Health Allied Sci [serial online] 2018 [cited 2019 Mar 22];7:250-5. Available from: http://www.ijhas.in/text.asp?2018/7/4/250/243266
| Introduction|| |
Pseudomonas aeruginosa is one of the opportunistic pathogens causing nosocomial infections such as burns, skin and soft tissue infections, pneumonia, urinary tract infections, septicemia, and bacteremia and lead to severe disease in immunocompromised patients.,
Till date, carbapenems have been found to be among the most effective drug for the Gram-negative bacteria for both fermenters and nonfermenters. With increasing development of carbapenemase resistance, these bacteria also develop multidrug resistance to the antibiotics such as β-lactam group, aminoglycosides, and fluoroquinolones. These multidrug-resistant strains are only susceptible to colistin, tigecycline, and polymyxin B.,
P. aeruginosa are the difficult bacteria to treat as they develop resistance even during the treatment course, one of the reasons may be biofilm formation. This altogether increases the morbidity and mortality, prolonging the hospital stay, and cost due to the dearth of treatment options.,
There have been various mechanisms responsible for carbapenem resistance in P. aeruginosa such as carbapenemase enzyme production, decreased bacterial outer membrane permeability, and constitutive expression of efflux pump. The carbapenemase enzymes responsible are metallo-beta-lactamases (MBLs) such as IMP, VIM, NDM, and SPM and among the non-MBL enzymes are KPC, OXA, and GES types.
NDM-1 gene has been most commonly isolated from Enterobacteriacae members but it is also recovered from nonfermenters such as Pseudomonas and Acinetobacter causing both community-acquired as well as hospital-acquired infections. Although the Prevalence of bla NDM-1 in P. aeruginosa is not that high compared to the fermenters, we have tried to highlight its prevalence and antimicrobial resistance pattern in our hospital setup.
| Materials and Methods|| |
Collection of isolates
We have taken 460 nonduplicate P. aeruginosa which were obtained from various clinical samples including pus, sputum, various body fluids, wound swab, urine, and blood, received from inpatient departments of the hospital during June 2014 to December 2014 were included in the study. The study was performed after the ethical committee approval.
Microbiological processing of clinical samples
All the clinical samples were received in the bacteriology laboratory of the microbiology department and processed. Pus, sputum, various body fluids collected in a sterile container, and wound swab were inoculated on sheep blood agar and MacConkey agar. Urine samples collected in a sterile universal container were inoculated on cysteine–lactose–electrolyte-deficient agar media. Blood samples collected in brain–heart infusion broth in a ratio of 1:5–1:10 (blood to broth) were incubated overnight at 37°C and then subcultured on sheep blood agar and MacConkey agar. Inoculated media were incubated aerobically at 37°C for 16–24 h.
Identification of Pseudomonas aeruginosa
The culture plates were examined for the growth after incubation and were processed further for identification. P. aeruginosa was identified on the basis of colony morphology, pigment production, Gram's staining, catalase, oxidase, standard biochemical tests, and growth on cetrimide agar.
Phenotypic detection of carbapenemase production
Isolates were first screened for the production of carbapenemase. The screening was done by disk diffusion method using imipenem 10 μg disk (HiMedia). A lawn culture of 0.5 McFarland standard suspension prepared from the isolate to be tested was done on Mueller–Hinton agar (MHA, HiMedia), and imipenem disk was applied on the surface of agar and incubated aerobically at 37°C for 16–18 h. The zone of inhibition ≤18 mm was considered indicative of carbapenemase production as per the Clinical and Laboratory Standard Institute (CLSI) 2014 guidelines. Quality control strain used for the test was P. aeruginosa ATCC 27853 and Escherichia More Details coli ATCC 35218 (for β-lactam/β-lactamase inhibitor combinations).
Phenotypic confirmatory test
Imipenem-ethylenediaminetetraacetic acid (EDTA) double-disk synergy test was used to determine carbapenemase production in P. aeruginosa. A 0.5 McFarland suspension of test isolate was inoculated in an MHA plate and two imipenem 10 μg disks were placed on the surface of agar. About 5 μl of 750 μg EDTA solution was applied over one imipenem disk. After 16–18 h of aerobic incubation at 37°C, the plate was examined for the zone of inhibition around imipenem and imipenem-EDTA disks. A difference in the zone of inhibition >7 mm between the two disks was considered as carbapenemase producer.
Antimicrobial susceptibility testing of carbapenemase producer
Antimicrobial susceptibility testing of all the isolates showing carbapenemase production by phenotypic method was determined by Kirby–Bauer disk diffusion method on MHA as per the CLSI 2014 guidelines. The antibiotics used were (potency in μg/disk, HiMedia) ceftazidime (30), gentamicin (10), tobramycin (10), piperacillin (100), ticarcillin (75), amikacin (10), aztreonam (30), cefepime (30), piperacillin-tazobactam (100/10), ticarcillin-clavulanate (75/10), ciprofloxacin (5), levofloxacin (5), norfloxacin (10), doripenem (10), imipenem (10), meropenem (10), colistin (10), and Polymyxin B (300 units).
Detection of New Delhi metallo-β-lactamase-1 gene by polymerase chain reaction
DNA was extracted from the isolates by Real Genomics DNA Extraction kit (version 2013-1, RBC Bioscience Ltd.). Polymerase chain reaction (PCR) of the extracted DNA was performed to determine the New Delhi metallo-β-lactamase-1 (bla NDM-1) gene in the carbapenemase-producing P. aeruginosa isolates. Primers used for amplification of the gene which amplified an internal fragment of 621 bp of the bla NDM-1 gene as depicted in [Table 1]. The amplification of DNA was performed in Applied Biosystems thermocycler with a final volume of 25 μl. The PCR conditions for the amplification of gene were denaturation at 94°C for 10 min, 36 cycles of amplification at 94°C for 30 sec, 54°C for 40 s, and 72°C for 50 s and final extension at 72°C for 5 min. The amplified DNA products were visualized by Gel Electrophoresis using 1% (w/v) agarose gel in 1x TBE (1.0M Tris/0.9M Boric acid/0.01M EDTA) with 0.05 mg/lt ethidium bromide [Figure 1].
|Table 1: Primers for New Delhi metallo-β-lactamase-1 gene amplification in Pseudomonas aeruginosa|
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|Figure 1: Gel electrophoresis of New Delhi metallo-β-lactamase-1 in Pseudomonas aeruginosa|
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This was a prospective cross-sectional study conducted in the Department of Microbiology, Hind Institute of Medical Sciences, Barabanki, Uttar Pradesh, India. Ethical committee approval was taken before the study and informed consent was obtained from patients.
The results were recorded in a Microsoft Excel sheet and were statistically analyzed. Chi-square test was used to analyze the data. P < 0.01 was considered as statistically significant.
| Results|| |
A total of 460 isolates of P. aeruginosa were obtained from various clinical samples such as urine (258), sputum (95), blood (43), pus (39), and body fluids (25). Among the different clinical samples, carbapenem-resistant P. aeruginosa (CRPA) was mostly found in blood samples (41%; 18/43), followed by other samples which was statistically significant (P < 0.01) [Table 2].
|Table 2: Prevalence of carbapenem-resistant Pseudomonas aeruginosa and carbapenem-sensitive Pseudomonas aeruginosa among different clinical samples#|
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The present study showed 74 CRPA among 460 samples constituting 16.08%. All the CRPA isolates were subjected to IMP-EDTA double-disk synergy test for MBL detection, where 61 isolates were positive with the test. Out of these, 18 isolates were bla NDM-1 positive [Table 3].
|Table 3: Distribution of carbapenemase-producing Pseudomonas aeruginosa isolates|
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We have performed antibiotic sensitivity by Kirby–Bauer disk diffusion method, which showed that carbapenemase-producing isolates were completely resistant to ceftazidime (100%) and aztreonam (100%), above 50% resistant to β-lactams such as piperacillin (97.3%), ticarcillin (95.9%), ticarcillin-clavulanate (68.9%), cefepime (58.1%), and piperacillin-tazobactam (55.4%). Among aminoglycosides, there was higher resistance to gentamicin (79.7%) compared to tobramycin (56.7%) and amikacin (54%). Fluoroquinolones were highly resistant such as ciprofloxacin (91.1%), levofloxacin (77%), and norfloxacin (82.4%) in urine isolates. All the isolates were completely sensitive to colistin and polymyxin B [Figure 2].
|Figure 2: Antibiotic resistance pattern among Pseudomonas aeruginosa isolates|
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| Discussion|| |
Carbapenem resistance is emerging rapidly in nonfermenters such as P. aeruginosa. In our study, we have seen 16.08% (74/460) CRPA isolates. In South India, 12% carbapenem resistance was reported in Karnataka and 28.8% in Chennai in a study of Shanthi et al. A study in Taiwan showed 10% CRPA, that is, 349 out of 3408 samples.
P. aeruginosa is one of the most common causes of hospital-acquired infections; hence, it is important to identify these CRPA from different sources to optimize treatment and reduce the morbidity and mortality. The prevalence of CRPA was highest in bloodstream infections (41.8%; n = 18/43), followed by other samples. This was statistically significant with P < 0.01. In a study by Meradji et al., infections caused by P. aeruginosa were the skin, surgical site, and soft-tissue infection (70%), followed by lower respiratory tract infection (17.5%), urinary tract infection (11.25%), and bloodstream infection (1.25%) as per the anatomical location.
We have found that three patients expired among the CRPA cases followed, of which, two cases were bla NDM-1 positive. The mortality rate was 0.6% (3/460) among the patients infected with P. aeruginosa during the study period.
NDM-1 gene was first identified in Klebsiella spp. in India in 2009, later in other members of Enterobacteriaceae. NDM-1 gene was first reported in 2011 in P. aeruginosa in Serbia, till that time no study was reported on NDM-1 in P. aeruginosa. In 2013, both Shanthi et al. from South India and Khajuria et al. from Pune reported four NDM-1 genes positive in P. aeruginosa., Subsequently, from Italy in the same year 2013, NDM-1 gene in P. aeruginosa was first reported, patient was previously hospitalized in Serbia. Then, in 2014, two cases of NDM-1 in P. aeruginosa were detected in Egypt, they had no history of travel to India or Europe. In Singapore, the first case was reported from Southeast Asia in 2015.
Nowadays, MBL-producing isolates are increasing worldwide and are commonly associated with infections in hospital setup. The present study has reported 13.2% (61/460) MBL production in P. aeruginosa isolates. A study by Navaneeth et al. reported 12% MBL in P. aeruginosa. Out of 74 CRPA, our study reported 61 MBL using IMP-EDTA disk test. We have not performed modified Hodge test (MHT) as the CLSI does not recommend the use of it for P. aeruginosa. However, some studies like Shanthi et al. have performed MHT using meropenem disk.,, MBL detection from CRPA in our study was 82.4% (n = 61/74) which is close to 90% (n = 110/122) from imipenem-resistant P. aeruginosa in a study from Iran and higher than Polotto et al., a Brazilian study which has detected 31.4% (n = 17/54) MBL from imipenem-resistant P. aeruginosa., A study by Irfan et al. in Pakistan showed 100% MBL positive by IMP-EDTA disk test in imipenem-resistant P. aeruginosa.
In the present study, the overall prevalence of bla NDM-1 isolates was 3.91% (n = 18/460) from all P. aeruginosa isolates which is concordant to Khajuria et al. with 2% prevalence (n = 4/200) and Shanthi et al. with 1.87% (n = 4/212) in South India from all P. aeruginosa isolates., The prevalence of bla NDM-1 among CRPA isolates was 24.32% (n = 18/74) in our study which was higher than Khajuria et al. 10% (n = 4/40) and Shanthi et al. 6.5% (n = 4/61) among CRPA isolates.,
The present study documented 18 bla NDM-1 among 61 MBL-producing P. aeruginosa. A total of 43 MBL-producing isolates which were negative for bla NDM-1 may have other genes present such as bla VIM, bla IMP, or bla SIM. We have limited our study to bla NDM-1 detection using conventional PCR therefore, we cannot comment on other genes. As reported from various studies in India, bla VIM is the most common MBL gene among P.aeruginosa isolates followed by other genes including bla NDM-1., Amudhan et al. have reported 55.7% bla VIM and 3.3% bla IMP gene among carbapenem resistant P.aeruginosa isolates and another study showed 13.3% bla VIM and 1.3% bla IMP from multidrug resistant P.aeruginosa.
Although the prevalence of bla NDM-1 is relatively low in Pseudomonas, still this figure is important due to multidrug resistance among these isolates. Along with β-lactams, the CRPA strains are also resistant to other groups of antibiotics such as aminoglycosides and fluoroquinolones. This study has noticed that resistance pattern in these CRPA is higher as compared to carbapenem-sensitive P. aeruginosa isolates. These strains are completely sensitive to colistin and polymyxin B in our study similar to the study by Ramakrishnan et al. from Puducherry. In addition, Carattoli et al. and Lin et al. showed 100% sensitivity to colistin, whereas Shanthi et al. showed 100% sensitivity to polymyxin B.,, Tigecycline, in effective in Acinetobacter but not in Pseudomonas. Hence, we are left with limited antibiotics of choice for treatment of CRPA.
| Conclusion|| |
Due to increasing multidrug resistance in P. aeruginosa and its nosocomial nature, it is becoming a great threat to the world of antimicrobials. Although we have colistin and polymyxin B sensitivity to CRPA, these can never be the first-line drugs due to toxicity; therefore, we have to judiciously use the antibiotics to prevent drug resistance and should make an effort to generate an antibiotic policy in the hospital which will help the clinicians to choose the appropriate drug for empirical treatment.
There are tests available such as carbaNP test which can be used to detect carbapenemase production at places where the molecular diagnostic facilities are not available. It is a simple and rapid test which can be performed even by laboratory technicians and can help in infection control. The need of hour is to timely identify the drug-resistant pathogens and treat them with proper antibiotics effectively to reduce the morbidity and mortality and also to prevent their spread in the community.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]