PHENOTYPIC AND MOLECULAR DETECTION OF CARBAPENEMASE NEW DELHI METALLO BETA LACTAMASE-1 (NDM-1) GENE AMONG PSEUDOMONAS AERUGINOSA FROM VARIOUS CLINICAL ISOLATES

PHENOTYPIC AND MOLECULAR DETECTION OF CARBAPENEMASE NEW DELHI METALLO BETA LACTAMASE-1 (NDM-1) GENE AMONG PSEUDOMONAS AERUGINOSA FROM VARIOUS CLINICAL ISOLATES

Sheeba Ali Siddiqui ^{* 1}, C. M. Noorjahan ¹ and N. Arunagirinathan ²

P.G. & Research Department of Zoology ¹, Justice Basheer Ahmed Sayeed College For Women, Teynampet, University of Madras, Chennai - 600018, Tamil Nadu, India.

Department of Microbiology ², Presidency College, University of Madras, Chennai - 600005, Tamil Nadu, India.

ABSTRACT: Pseudomonas aeruginosa is an opportunistic pathogen which acquires resistance to antibiotics such as carbapenems and cefepime. Information regarding (NDM-1) gene producing Pseudomonas aeruginosa is available, but data regarding their degree of infection and percentage in hospital settings are scarce. Hence, this study was carried out to determine the occurrence of blaNDM-1 gene among clinical isolates of multidrug-resistant Pseudomonas aeruginosa in a tertiary care hospital in Chennai, Tamil Nadu, India. A total of 126 isolates of P. aeruginosa isolated from various clinical samples were evaluated for carbapenem resistance and MBL production. In the present study it was found that majority of isolates (39.7%) were resistant to imipenem followed by ertapenem (35.9%) and doripenem (24.3%). A total of 31/78 (39.7%) isolates of P. aeruginosa were resistant to one or both carbapenems (imipenem and ertapenem) used in the screening test. Out of 78 isolates, 27 (34.6%) and 25 (32.1%) isolates were MHT positive and MBL producers by Modified Hodge Test (MHT) and MBL E-Strip test respectively and Out of 25 MBL positive isolates, 7 (28%) isolates were positive, and 18 (72%) were negative for NDM-1 gene production. In this study prevalence of plasmid encoding NDM-1 gene was noted in Multidrug-resistant Pseudomonas aeruginosa. Screening of Pseudomonas aeruginosa along with routine antimicrobial susceptibility testing, Phenotypic and molecular screening should also be carried out regularly to reflect the proper number of metallo-beta-lactamase producers.

NDM-1 Gene, Combined disc test, E-test, Metallo beta lactamases, Modified Hodge test, Pseudomonas aeruginosa

INTRODUCTION: Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in patients. Carbapenems, especially imipenem, are used for the treatment of these infections.

The prevalence of imipenem resistance P. aeruginosa has been increasing worldwide. Resistance to carbapenems is due to impermeability via the loss of the OprD porin, the up-regulation of an active efflux pump system of the cytoplasmic membrane, or the production of metallo-β-lactamases (MBLs).

These mechanisms can lead to treatment failure in carbapenem therapy of P. aeruginosa infections ¹. Resistance to antibiotics has increased over the years among Pseudomonas aeruginosa as nearly most of the strains are now resistant to all commonly used antibiotics. These bacteria produce Metallo-β-lactamases (MBLs). Multidrug resistance among these organisms makes the treatment of infections caused by the very expensive and difficult to treat ². Metallo-β-lactamases (MBLs) are metalloenzymes of Ambler class B and are clavulanic acid-resistant enzymes. They require divalent cations of zinc as co-factors for enzymatic activity and are universally inhibited by ethylenediamine tetra-acetic acid (EDTA), as well as other chelating agents of divalent cations ³. Metallo-β-lactamases producing P. aeruginosa isolates were first reported in Japan in 1991 and since then have been detected in various countries ⁴.  Carbapenem groups of antibiotics play an important role in managing gram negative nosocomial infections because of their broad-spectrum activity and stability against hydrolysis by most of the β-lactamases, including extended-spectrum β-lactamases (ESBLs) ⁵.

The most potent antibacterial agents used for the treatment of infections caused by multidrug-resistant gram-negative bacilli are carbapenems, including imipenem (IPM or IP) and meropenem ⁶. Carbapenems are now frequently used as a reversed drug in treating infections caused by multidrug-resistant gram-negative bacilli. The detection of carbapenemase is difficult.  It can be detected by phenotypic as well as genotypic methods. Among phenotypic tests   Modified Hodge Test (MHT) is a relatively easy and simple method to be performed in a laboratory ⁷.

Many multidrug-resistant bacteria produce New Delhi metallo β lactamase-1, a type of carbapenemase which inactivates all β-lactams except aztreonam ³. Bacterial plasmids encoding the blaNDM-1 gene also encodes for other genes responsible for causing resistance to all amino-glycosides, macrolides, and sulphamethoxazole, thus making these isolates multidrug-resistant or resistant in some cases to all antibiotics ⁸. NDM-1 gene producing organisms may spread the multiple drug resistance to non-resistant organism through horizontal gene transfer. The presence of such asymptomatic carriers bearing multidrug-resistant NDM-1 gene organisms in the hospital environment is an alarming situation ⁹. Hence this study was carried out for Phenotypic and molecular detection of carbapenemase New Delhi Metallo beta Lactamase-1 (NDM-1) gene among Pseudomonas aeruginosa from various clinical isolates and to evaluate different phenotypic methods to guide clinicians in prescribing a proper antibiotic and controlling nosocomial infections.

MATERIALS AND METHODS:

Clinical Isolates: The samples were collected from a tertiary care hospital in Chennai, and the study was carried out for a period of one year from November 2017 to October 2018. A total of 126 isolates of P. aeruginosa isolated from various clinical samples were evaluated for carbapenem resistance, MBL production, and molecular detection of the NDM-1 gene. The isolates were identified by standard laboratory techniques. Subculturing was done on a regular basis in order to maintain the fresh cultures for the experiment. P. aeruginosa ATCC 27853 was included as a quality control strain ¹⁰. The isolates were confirmed by Grams staining, biochemical tests, pigment production, and growth at 42 °C ¹⁰. Antibiotic susceptibility testing was done on Mueller–Hinton agar by Kirby–Bauer disc-diffusion method, and the results were interpreted as per the CLSI ¹¹. Screening for MBL production was done by Kirby–Bauer disc diffusion method ¹², Imipenem-EDTA Combined Disc Test (CDT), and Modified Hodge Test (MHT) ^{13, 14}. Confirmation of MBL production was done by the E-test (Himedia Mumbai, India), and molecular detection of the NDM-1 gene was carried out by PCR ^{12, 15}.

Screening of Isolates for MBL Production: The in-vitro antibiotic cultural sensitivity of P. aeruginosa for MBL production was done by the disc diffusion method ¹¹. The following antibiotics were used in the screening test: imipenem (10 µg), doripenem (10 µg), and ertapenem (10 µg) (Himedia Mumbai, India). The size of the zone of inhibition was read after overnight incubation at 37 °C and interpreted according to recommended MBL screening criteria as specified in CLSI protocol, M100-S22 ¹².

Confirmatory Methods:

Imipenem-EDTA Combined Disc Test (CDT): MBL production was confirmed by IMP-EDTA combined disc test ¹³. Imipenem disc (10 µg) and Imipenem/EDTA disc (10 µg/750 µg) (Himedia Mumbai, India) were placed on the dried plate inoculated with the P. aeruginosa. The disc was placed 10 mm apart from edge to edge. After 24 h of incubation at 37 °C, the inhibition zones of IMP and IMP/EDTA discs were compared. For MBL producing organisms, discs with IMP/EDTA increased inhibition zones by 8 to 15 mm, while the increase of such zones for MBL negative isolates were 1 to 5 mm according to CLSI guidelines, 2013 ¹².

Modified Hodge Test: The Meropenem and Imipenem resistant strains were subjected to MHT for detection of Carbapenemases. An overnight culture suspension of E. coli ATCC 25922, which was adjusted to 1:10 dilution of the 0.5 McFarland standard, was inoculated on the surface of the Muller Hinton agar plate evenly. After the brief drying at room temperature, 10 µg Ertapenem disk was placed in the center of the plate. Imipenem resistant test strain from an overnight culture was streaked heavily from the edge of the disk to the edge of the plate. After 24 h the presence of a distorted or clover leaf-shaped inhibition zone was interpreted as MHT positive due to Carbapenemase production by the test strain ¹⁴.

MBL E – Test: MRP/MRP+EDTA E -test strips (Himedia Mumbai, India) consisted of Meropenem (MRP) (4-256 µg/ml) and MRP (1-64 µg/ml) plus a constant level of EDTA. An inoculum (0.5 Mc Farland standards) was prepared from 24 h old culture of the P. aeruginosa test strain, inoculated on Muller Hinton Agar plate with the help of sterile cotton swabs.

After brief drying, E test strips were applied on MHA plates and incubated for 18-24 h at 37 °C. The MIC endpoints were read where the inhibition ellipses intersected the strips. A ratio of MICs of the Meropenem (MRP) to MRP+EDTA of ≥ 8 was interpreted as MBL positive ¹². S. maltophilia ATCC 13636 and P. aeruginosa ATCC 27853 were used as positive and negative control strains, respectively, in the MBL E- test.

Molecular Detection of NDM-1 Gene: All 25 isolates, which were confirmed phenotypically to be positive for MBL production by MBL E-Test, were subjected to PCR using group-specific primers to detect the NDM-1 gene. The primers used in the study are shown in Table 4.

DNA was extracted by boiling lysis method. A single colony was inoculated into 1 ml Luria bertani broth and incubated at 37 °C for 24 h. It was subjected to centrifugation at 10,000 rpm, 25 °C for 10 min. The pellet obtained was suspended with 500 µl milli-Q water and boiled at 100 °C for 10 min. It was then stored overnight at -20 °C. The next day it was subjected to centrifugation at 10,000 rpm, 25 °C for 10 min.

The supernatant was collected and used as the template for the PCR. The cycling conditions were as follows: denaturation at 94 °C for 10 min, followed by 35 amplification cycles (94 °C for 30 sec, 58.5 °C for 40 sec, 72 °C for 50 sec) and a final extension cycle (72 °C for 5 min). The DNA band patterns were evaluated by electrophoresis with 1.5% agarose gel in 1X TBE buffer. A 100 bp ladder molecular weight marker was used to measure the molecular weights of tested amplified products. The presence of bands of molecular weight of 621bp suggests the presence of the NDM-1 gene on gel electrophoresis ¹⁵.

Results:

Clinical Isolates: The results of the clinical isolates are presented in Table 1. A total of 126 clinical isolates were collected from tertiary care hospitals in Chennai, out of which 78 Multidrug-resistant isolates were screened for Carbapenemase and MBL production.

Phenotypic Detection of MBL Producers and their Confirmation:

Screening of Isolates for MBL Production, Imipenem-EDTA Combined Disc Test (CDT), Modified Hodge Test, MBL E – Test: The results of in-vitro antibiotic cultural sensitivity to different carbapenems are presented in Table 2. Majority of isolates (39.7%) were resistant to imipenem followed by ertapenem (35.9%). However, lower proportions of isolates were recorded resistant to doripenem (24.3%) in Table 2 / Fig. 1.

A total of 31/78 (39.7%) isolates of P. aeruginosa were resistant to one or both carbapenems (imipenem and ertapenem) used in the screening test. The Imipenem resistant isolates 31 (39.7%) exhibited a zone size enhancement of ≥ 7 mm in the combined disc test Table 3/Fig. 1. Majority (60.3%) isolates were non-MBL producers.

Out of 78 isolates, 27 (34.6%) and 25 (32.1%) isolates were MHT positive and MBL producers by Modified Hodge Test (MHT) and MBL E-Strip test respectively Table 3/Fig. 2 & 3. S. maltophilia ATCC 13636 and P. aeruginosa ATCC 27853 were used as positive and negative quality control strains.

Molecular Detection of NDM-1 Gene: The results of the molecular detection of the NDM-1 gene are presented in Table 5 /Fig. 4. PCR for detection of NDM-1 was carried out for 25 isolates. Out of 25 isolates, 7 (28%) isolates were positive, and 18 (72%) were negative for NDM-1 gene production.

TABLE 1: ISOLATION OF PSEUDOMONAS AERUGINOSA FROM VARIOUS CLINICAL ISOLATES

TABLE 2: IN-VITRO CULTURAL SENSITIVITY ASSAY USING CARBAPENEMS AGAINST P. AERUGINOSA ISOLATES

FIG. 1: ISOLATES SHOWING RESISTANCE TOWARDS CARBAPENEMS LEFT SIDE OF THE PLATE (IMIPENEM, ERTAPENEM, DORIPENEM). RIGHT SIDE OF THE PLATE (IMIPENEM -IMIPENEM +EDTA) IS POSITIVE FOR MBL BY PHENOTYPIC TEST (COMBINED DISC TEST)

FIG. 2: THE PRESENCE OF A DISTORTED OR CLOVER LEAF SHAPED INHIBITION ZONE IS A MHT POSITIVE DUE TO CARBAPENEMASE PRODUCTION BY THE TEST STRAIN

FIG. 3: A RATIO OF MICS OF THE MEROPENEM (MRP) TO MRP+EDTA OF ≥ 8 WAS INTERPRETED AS MBL POSITIVE

TABLE 3: PHENOTYPIC DETECTION OF METALLO-Β-LACTAMASE PRODUCING P. AERUGINOSA ISOLATES

TABLE 4: PRIMER SEQUENCE

TABLE 5: MOLECULAR DETECTION OF NDM-1 GENE

FIG. 4: AGAROSE GEL ELECTROPHORESIS (1.5%) REPRESENTS 621 BP BLANDM-1 GENE IN PSEUDOMONAS AERUGINOSA., LANE 1: 100BP DNA LADDER, LANE 2: POSITIVE CONTROL, LANE 3: NEGATIVE CONTROL, LANES 4 & 12: NEGATIVE ISOLATES FOR NDM-1 PRODUCTION (AMPLICON SIZE-621 BP); LANES 5-11: POSITIVE ISOLATES FOR NDM-1 PRODUCTION (AMPLICON SIZE-621 BP)

DISCUSSION: Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infection in patients with weakened immune systems and it has become the important cause of nosocomial infections with a high mortality rate ^{16, 17}.

It is predominantly responsible for emerging hospital-acquired infections and poses serious health concerns due to increased levels of multidrug resistance ¹⁸. P. aeruginosa is resistant to many classes of antimicrobial agents and can acquire resistance by mutation and horizontal transfer of resistance determinants. The risk of mortality and morbidity in infections caused by P. aeruginosa is increased due to wrong or delayed initial antibiotic therapy, especially when the infection is caused by multidrug-resistant pathogens ^{19, 20}.

In our study total of 126 P. aeruginosa isolates were isolated from the following clinical samples  Pus 43(34.1%), Wound Swab 21(16.6%), Cerebrospinal fluid (CSF) 2 (1.6%), Bronchoalveolar Lavage (BAL) 4 (3.1%), Endotracheal Aspirate (ETA) 7(5.6%), Sputum 15 (12%), Blood 9 (7.1%), Ear swab 13(10.3%), Urine 7(5.6%), High Vaginal Swab (HVS) 5 (4%). The highest numbers of isolates were obtained from the pus sample and the lowest from CSF. In a similar study carried out previously, 110 different organisms were isolated from a total of 170 clinical samples such as pus, urine blood, sputum, and drain fluid, and they reported 15.78% of isolates were P. aeruginosa and 57 (22.4%) isolates were isolated from 254 pus samples in a study carried out in Bathinda ^{21, 22}. The occurrence of multidrug-resistant Metallo-β-lactamase P. aeruginosa isolates in a hospital setting is of concern as it poses a problem in therapy and infection control management. In our study, the prevalence of MBL-PA isolates was 39.7%.

Metallo-β-lactamase producing Pseudomonas aeruginosa is an important nosocomial pathogen that shows resistance to all β-lactam antibiotics except monobactam. In another study, they isolated 121 (96.1%) Pseudomonas aeruginosa out of 126 clinical isolates from the following samples, pus 35 (27.8%), urine 25 (19.84%), endotracheal aspirate 24 (19.04%), blood 14 (11.11%), and sputum 4 (3.17%) ²³. Similarly, 21% of MBL positive P. aeruginosa were isolated from urine, wound swabs, sputum, blood, tissue aspirates, ear swab, and CSF samples in a study carried out in Sudan ²⁴. While in a previous study in Jaipur, it was reported 20% ²⁵. 18.37% isolates of P. aeruginosa were positive for MBL production from Uttarakhand ²⁶. 41.3% of Pseudomonas aeruginosa isolates were screened positive for MBL production, which was reported from Bengaluru ²⁷.

Similar to our study, the prevalence of MBL production was found to be 10% in India, 53.2% in Iran ²⁸. 12% in Canada, 12.7% in the United Arab Emirates, 13.4% in Russia, 14% in Spain, 38.3% in São Luis of Brazil, 47.3% in Taiwan, and 62% in Greece 29,30. 93.75% isolates were MBL positive in a study carried out in Karim Nagar, and 20.3% were positive for MBL production in a study carried out in 2019 in Thailand ^{31, 32}. In another study, 13.2% of isolates in Barabanki and 52% of isolates in Bahrain were found to be positive for MBL production ^{33, 34}. Similarly, ³⁹ isolates were screened by the combined disc and disc diffusion methods, and 64.1% were found to be MBL producers in Egypt ³⁵. MBL was detected by a modified Hodge, and imipenem-EDTA double-disk synergy test and 14.4% isolates were positive for MBL production in Aligarh ³⁶.

Resistance to carbapenem antibiotics in various Gram-negative bacteria suggests that they may be responsible for increased mortality. The emergence of resistance was associated with a significantly increased number of adverse outcomes ³⁷. Carbapenems are the most effective antimicrobial agents against gram-positive and gram-negative bacteria, including P. aeruginosa. Carbapenems have the beta-lactam ring, and, like all other beta-lactams, they inhibit bacterial cell wall synthesis by binding to and inactivating Penicillin Binding Proteins (PBPs). This unique molecular structure offers them their exceptional stability to many beta-lactamases, including AmpC and most of the extended-spectrum beta-lactamases (ESBLs) ³⁸. Pseudomonas aeruginosa isolates acquire resistance to carbapenems via several mechanisms including overexpression of efflux systems, change or lack of outer membrane proteins (such as OprD porin), chromosomal AmpC beta-lactamase, and production of carbapenemases ³⁹.

A simple screening method using a combined disc diffusion test has been very useful to screen MBL positive isolates. We screened MBL producers among imipenem resistant isolates by three methods, the Combined Disc Test (CDT), Modified Hodge test (MHT), and E - strip test for MBL screening and found that majority of isolates (39.7%) were resistant to imipenem followed by ertapenem (35.9%) and doripenem (24.3%). A total of 31/78 (39.7%) isolates of P. aeruginosa were resistant to one or both carbapenems (imipenem and ertapenem) used in the screening test. The Imipenem resistant isolates 31 (39.7%) exhibited a zone size enhancement of ≥ 7mm in the combined disc test.

The majority (60.3%) isolates were non-MBL producers. Out of 78 isolates, 27 (34.6%) and 25 (32.1%) isolates were MHT positive and MBL producers by Modified Hodge Test (MHT) and MBL E-Strip test, respectively. Based on another study carried out previously for the screening of MBL production, 66 isolates were screened positive. The prevalence of MBL producing isolates of P. aeruginosa was 15% (24/160) based on the E-test result. MHT showed the highest sensitivity (87.5%), followed by CDT (79.2%), while specificity was highest for DDST (100%), followed by PT (95.2%). Out of 24 MBL producers, 15 isolates (62.5%) were resistant to both imipenem (IPM) and meropenem ²⁷. It is lower in incidence as compared to our study. Similarly, 94.6% of carbapenemase production in P. aeruginosa isolates were reported in Delhi ²³. In a previous study, 28.17% of isolates in Iran and 16.08% of isolates in Barabanki were found to be carbapenem resistant P. aeruginosa ^{40, 33}. While in a previous study in Egypt, it was reported 26.5% 35. In a retrospective cohort study carried out in Pittsburgh Medical Centre, they reported a 19% mortality rate in patients with bacteremia is due to carbapenem-resistant (CR) Pseudomonas aeruginosa ⁴¹. In this study, out of 25 isolates, 7 (28%) isolates were positive, and 18 (72%) were negative for the NDM-1 gene. Similarly, 46.06% isolates in Delhi, 3.91% isolates in Barabanki, 2.5% isolates in Bahrain, and 10.4% isolates in Singapore were found to be positive for the production of NDM-1 gene ^{23, 33, 34, 42}. In another study carried out in Iraq during 2018. They reported the emergence of Pseudomonas aeruginosa carrying NDM-1 gene variants, which exhibited resistance to imipenem and meropenem for the first time in Iraq ⁴³.

CONCLUSION: The prevalence of multidrug-resistant Pseudomonas aeruginosa infections leads to a worldwide increase in the occurrence of MBL-PA, which is alarming. A high rate of the NDM-1 gene producer was noted among Multidrug-resistant Pseudomonas aeruginosa. Apart from performing the only antimicrobial sensitivity tests, phenotypic and molecular screening should also be employed regularly to find out the actual number of the NDM-1 resistance gene and for proper diagnosis and management of all P. aeruginosa infections.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

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    How to cite this article:

    Siddiqui SA, Noorjahan CM and Arunagirinathan N: Phenotypic and molecular detection of carbapenemase New Delhi metallo beta lactamase-1 (NDM-1) gene among Pseudomonas aeruginosa from various clinical isolates. Int J Pharm Sci & Res 2020; 11(11): 5856-63. doi: 10.13040/IJPSR.0975-8232.11(11).5856-63.

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