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Prevalence of Carbapenem Resistant Gram Negative Bacilli Harboring blaNDM-1 Gene Isolated in a Tertiary Care Hospital



Manisa Sahu, Priyadharshini Sekar, Revathy Ramalingam, Pallavi Bhalekar, E. Suguna, Gnana Soundari Palani, Padma Krishnan and Godfred A. Menezes
 
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ABSTRACT

Background: Increase in antimicrobial resistance is of major concern worldwide. This is largely attributed to broad spectrum β-lactamase production. New Delhi Metallo β-lactamase-1 (NDM-1) is a recently identified type of metallo-β-lactamase which has been increasingly viewed as a potential threat to global health. Objective: The aim of this study was to perform molecular detection of blaNDM-1 gene to determine its occurrence among clinical isolates of Gram negative bacteria. Methodology: A total of 178 Gram negative bacilli isolated from different clinical samples including urine, tissue, sputum, blood, pus, endotracheal secretion (ET secretion), stool, pleural fluid, cup tip, Peripherally Inserted Central Catheter (PICC) tip, drain tube and bile were included in the study. The isolates were identified by Vitek 2 GN cards and antibiotic susceptibility testing was performed by using Vitek 2 AST-N280 and AST-N281 cards (bioMe’rieux, SA, France), as per manufacturer’s instructions. The isolates were stocked and used for further study. PCR to detect the presence of blaNDM-1 gene was performed with all the isolates. The ERIC-PCR was performed with 17 blaNDM-1 positive representative isolates. Of the 178 isolates, a remarkably high incidence of 29.8% blaNDM-1 gene was found. Of the 53 blaNDM-1 positive cases, 17 representative isolates were studied for clonal relatedness by ERIC-PCR. Results: It was found that Klebsiella pneumoniae and Acinetobacter baumannii had 2 and 1 clonally related clusters, respectively. Pseudomonas aeruginosa and Escherichia coli were clonally divergent. We suggest that the genotypic detection of NDM-1 along with routine antimicrobial susceptibility test should be performed in all health centers worldwide. The blaNDM-1 gene has "An alarming potential" to spread and diversify among bacterial populations. Conclusion: Hence early identification of cases of NDM-related infections and prevention of their spread by implementing screening, hygiene measures and the isolation of carriers is required.

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  How to cite this article:

Manisa Sahu, Priyadharshini Sekar, Revathy Ramalingam, Pallavi Bhalekar, E. Suguna, Gnana Soundari Palani, Padma Krishnan and Godfred A. Menezes, 2016. Prevalence of Carbapenem Resistant Gram Negative Bacilli Harboring blaNDM-1 Gene Isolated in a Tertiary Care Hospital. Current Research in Bacteriology, 9: 9-16.

DOI: 10.3923/crb.2016.9.16

URL: https://scialert.net/abstract/?doi=crb.2016.9.16
 
Received: March 17, 2016; Accepted: May 09, 2016; Published: June 15, 2016



INTRODUCTION

Increase in antimicrobial resistance during the past decade in Enterobacteriaceae has become a major concern worldwide. This is largely attributed to broad spectrum β-lactamase production (Menezes and Menezes, 2013). Further, the emergence and global spread of carbapenem-resistant Enterobacteriaceae (CRE) is of great distress (Zou et al., 2015). Among the metallo β-lactamase's (MBLs), Verona integron-encoded metallo-β-lactamase (VIM), imipenemase (IMP) and New Delhi metallo-β-lactamase (NDM) are the most prevalent types. The blaNDM-1 was originally identified in Klebsiella pneumoniae and Escherichia coli in 2008 in Sweden from a traveller returning from India (Yong et al., 2009). The blaNDM-1 encodes a broad-spectrum β-lactamase that inactivates all β-lactams except aztreonam (Shenoy et al., 2014). The gene blaNDM-1 is mainly located on a plasmid and hence can transcend the genus/family barrier with ease and spread across the world (Menezes and Menezes, 2013). Currently, there are 16 variants of blaNDM.

Appropriate and rapid detection of NDM-1 producers is critical in implementing infection control measures. To better control NDM-1 producers, it is essential to understand the mediating mechanisms and their molecular epidemiology. In this study, we performed the molecular detection of blaNDM-1 gene among carbapenem resistant Gram negative clinical isolates cultured from clinical samples in a hospital in Mumbai, India. The ERIC-PCR was performed to study the clonal relatedness of blaNDM-1 gene positive carbapenem resistant Gram Negative Bacilli (GNB).

MATERIALS AND METHODS

Place of study and study period: The study isolates were from S.L. Raheja Hospital, Mumbai, India cultured during March, 2013 to May, 2014.

Patient population: Both males and females attending OP and IP across all age groups were included in the study.

Samples: Bacterial isolates were obtained from urine, tissue, sputum, blood, pus, endotracheal secretion (ET secretion), stool, pleural fluid, cup tip, Peripherally Inserted Central Catheter (PICC) tip, drain tube and bile.

Clinical bacterial strains: A total of 178 GNB isolates resistant to carbapenem were included in the study.

Control strains: Escherichia coli ATCC 25922, Klebsiella pneumoniae BA 2146 were used as controls where required.

Bacterial isolation, identification and antibiotic sensitivity testing: The isolates were identified by Vitek 2 GN cards and antibiotic susceptibility testing was performed by using Vitek 2 AST-N280 and AST-N281 cards (bioMe’rieux, SA, France), as per manufacturer’s instructions. The isolates were stocked and used for further study. Tigecycline MIC breakpoints were as per European Committee on Antimicrobial Susceptibility Testing (EUCAST., 2011) clinical breakpoints. The MIC breakpoints for other antimicrobial agents were interpreted as per Clinical and Laboratory Standards Institute (CLSI., 2013) guideline. The cefoperazone MIC breakpoint used for cefoperazone/sulbactam was as described by Tunyapanit et al. (2014).

DNA extraction: For DNA extraction, a single bacterial colony from an overnight grown culture was suspended in 100 μL of sterile MilliQ water and boiled for 5 min. The suspension was centrifuged at 8,000 rpm for 10 min. The supernatant containing bacterial DNA was used as template for PCR (Harish and Menezes, 2015).

PCR amplification for the detection of blaNDM-1 gene: PCR amplification of blaNDM-1 gene was carried out by using primers as described in an earlier study (Nordmann et al., 2011). The primers used were NDM-Fm5׳-GGTTTGGCGATCTGGTTTTC-3' and NDM-Rm5'-CGGAATGGCTCATCACGATC-3', which amplified a 621 bp internal fragment of the blaNDM-1 gene. The DNA from known blaNDM-1 positive and negative isolates were used as controls. The PCR was performed in a final reaction volume of 25 μL, containing 10 pmol each of forward and reverse primers, 2 μL of template DNA, 0.5 μL of 25 mM dNTPs, 2.5 μL of 10X amplification buffer, 0.5 U of Taq DNA polymerase. The PCR program consisted of following thermal cycling conditions: Initial denaturation step at 94°C for 10 min, followed by 36 cycles of 94°C for 30, 52°C for 40 and 72°C for 50 sec, followed by a final elongation at 72°C for 5 min. The application was performed using eppendorf thermocycler. The PCR products were run on 2% agarose (HI Media, Mumbai, India) gel, containing 1X tris-borate-EDTA (TBE) buffer and detected by ethidium bromide (Sigma) at 100 V for 30 min. The amplified PCR products were documented using Alpha Gel Imager (Alpha Innotech, USA) and the PCR band of 621 bp was visualized (Fig. 1).

Table 1: Distribution of occurrence of blaNDM-1 gene among clinical samples/isolates
ET: Endotracheal secretion, PICC: Peripherally inserted central catheter

Fig. 1:
Electrophoresis gel image demonstrating blaNDM-1 gene, Lane 1: Molecular mass marker (100 bp DNA ladder), Lane 2: Positive control, Lane 4: Negative control, Lane 3, 6 and 8: Test sample-positive (621 bp PCR product), Lane 5 and 7: Test sample-negative

Enterobacterial Repetitive Intergenic Concensus-Polymerase Chain Reaction (ERIC-PCR): The ERIC-PCR was performed as described by Dalla-Costa et al. (1998). Primers that were used are ERIClR, 5'ATGTAAGCTCCTGGGG-ATTCAC3' and ERIC2, S'AAGTAAGTGACTGGGGT-GAGCG3'. The PCR was performed in a final reaction volume of 25 μL, containing 10 pmol each of forward and reverse primers, 2 μL of template DNA, 0.5 μL of 25 mM dNTPs, 2.5 μL of 10X amplification buffer, 2.5 U of Taq DNA polymerase. The PCR program consisted of following thermal cycling conditions: Initial denaturation step at 95°C for 5 min, followed by 35 cycles of 92°C for 45 sec, 52°C for 1 min and 70°C for 10 min, followed by a final elongation at 70°C for 20 min. The application was performed using eppendorf thermocycler. The PCR products were run on 2% agarose (HI Media, Mumbai, India) gel, containing 1X tris-borate-EDTA (TBE) buffer and detected by ethidium bromide (Sigma) at 50 V for 1 h. The amplified PCR products were documented using Alpha Gel Imager (Alpha Innotech, USA).

RESULTS

Bacterial isolation and identification: During the 15 months study period, a total of 178 Gram negative isolates resistant to carbapenem were cultured. The isolates comprised of Klebsiella pneumoniae (86), Pseudomonas aeruginosa (33), Acinetobacter baumannii (24), Escherichia coli (22), Enterobacter aerogenes (3), Enterobacter cloacae (3), Citrobacter freundii (2), Citrobacter Koseri (1), Alcaligenes faecalis (1) and Morganella morgani (1).

Antibiotic sensitivity testing and PCR for blaNDM-1 gene: Of the 178 carbapenem resistant isolates, 53 (29.8%) were found to be positive for the blaNDM-1 gene (Table 1) by PCR.

Table 2: Demographic details of the subjects and antibiogram of the isolates positive for NDM-1 (n = 53)

Abbreviations: PTO: Post therapy outcome, ND: Not detected, ET: Endotracheal, DFI: Diabetic foot infection, DM: Diabetes mellitus, HTN: Hypertension, CKD: Chronic kidney disease, UTI: Urinary tract infection, AKA: Above-knee amputation, IHD: Ischemic heart disease, CA: Carcinoma, BMCA: Buccal mucosa carcinoma, PRES: Posterior reversible encephalopathy syndrome, NRDS: Neonatal respiratory distress syndrome, MDS: Myelodysplastic syndrome, PVD: Peripheral vascular disease, COPD: Chronic obstructive pulmonary disease, GI: Gastrointestinal, S: Susceptible, I: Intermediate, R: Resistant, Cd: Clindamycin, Clari: Clarithromycin, Cef/taz: Cefepime/tazobactam, Pt: Piperacillin/tazobactam, Cefo/sul: Cefoperazone+sulbactam, Mero: Meropenem, Teico: Teicoplanin , Azithro: Azithromycin, Amik: Amikacin, Cefp: Cefpodoxime, Imi: Imipenem, Cfn: Ceftriaxone, Oflox: Ofloxacin, Ciplox: Ciprofloxacin, Lz: Linezolid, Metro: Metronidazole, Cefot: Cefotaxime, Cl: Colistin and Tgc: Tigecycline

The 53 blaNDM-1 positive isolates included, Acinetobacter baumannii (13); Pseudomonas aeruginosa (7); Escherichia coli (12); Klebsiella pneumoniae (19), Citrobacter freundii (1) and Alcaligenes faecalis (1). These 53 isolates were obtained from different clinical samples-urine (19), tissue (11), sputum (9), blood (1), pus (2), ET secretion (6), pleural fluid (2), drain fluid (1), cup tip (1) and PICC tip (1). The blaNDM-1 was positive among 19 of 53 (35.8%) urinary isolates. Of the 53 blaNDM-1 cases, 16 (30.2%) were females. None of the stool and bile isolates were found to be positive for blaNDM-1.

Among the 53 blaNDM-1 positive isolates, a single K. pneumoniae isolate from urine was found to be resistant to colistin and was responsible for the death of the patient. 13/53 isolates, (24.5%) were resistant to tigecycline of which 9 isolates were K. pneumoniae, 2 isolates were P. aeruginosa and one isolate each of E. coli and A. faecalis (Table 2). Of the 53 blaNDM-1 positive cases, 8 had succumbed to death. An isolate of blaNDM-1 positive E. coli was responsible for Neonatal Respiratory Distress Syndrome (NRDS) leading to death. Further, none of the Enterobacter aerogenes and Enterobacter cloacae was found positive for blaNDM-1 (Table 2).

ERIC-PCR: The ERIC-PCR was performed to study the clonal relatedness of blaNDM-1 positive Gram negative isolates. This was performed for 17 representative isolates, details of which are in Table 3 and Fig. 2. Manual typing of the isolates revealed that 6 out of 9 blaNDM-1 positive K. pneumoniae isolates belonged to two clonal cluster types i.e., 2 isolates were of clonal cluster type 1, 4 isolates were of clonal cluster type 2. Two isolates of Acinetobacter baumannii were also clonally related. None of the other isolates i.e., 3 isolates of P. aeruginosa and 3 isolates of E. coli shared any clonal relatedness.

DISCUSSION

The NDM-1 producing bacteria are the most frequent cause of urinary tract infections (UTIs). They can also cause wound infections, bloodstream infections and pneumonia (Menezes and Menezes, 2013). In this study, among the blaNDM-1 positive cases, majority [19 (35.8%)] were UTI cases followed by wound infections [08 (22.9%)]. Of the 53 blaNDM-1 cases, 16 (30.2%) were females, whereas 37 (69.8%) were males. The higher rate of NDM-1 positivity was similar to the study by Shenoy et al. (2014). In this study, age ranged from 44-86 years except for one case of neonate. Our study indicates a high incidence (29.8%) of blaNDM-1. The reports of the incidence of NDM-1 has been highly variable. A recent study from China reported an incidence of 33.3% of NDM-1 among carbapenem resistant isolates (Qin et al., 2014) similar to present study. The 53 blaNDM-1 positive isolates cultured in current study included, A. baumannii (13), K. pneumoniae (19), E. coli (12), P. aeruginosa (7), A. faecalis (1) and C. freundii (1). The finding was corresponding with the findings of Shenoy et al. (2014). The NDM-1 producing A. baumannii can lead to outbreak of infections (Decousser et al., 2013).

Variable carbapenem resistance has been reported in NDM-1 positive isolates (Shenoy et al., 2014). Of the blaNDM-1 positive isolates in present study, a single isolate was found resistant to colistin. Among the isolates, 13 (24.5%) were found resistant to tigecycline. Except for few, most of the NDM-1 producers have been reported to remain susceptible only to colistin and tigecycline. Nevertheless, a high rate of tigecycline resistance (43.2%) has been reported (Shenoy et al., 2014). Due to limited therapeutic options, treatment of infections caused by NDM-1 producing pathogens is a major challenge for clinicians.

Fig. 2:
DNA fingerprints of isolates generated by ERIC-PCR and separated in a 2% (w/v) agarose gel, 1: K. pneumoniae, 2: K. pneumoniae, 3: P. aeruginosa, 4: K. pneumoniae, 5: P. aeruginosa, 6: K. pneumoniae, 7: K. pneumoniae, 8: K. pneumoniae, 9: K. pneumoniae, 10: E. coli, 11: A. baumannii, 12: K. pneumoniae, 13: E. coli, 14: K. pneumoniae, 15: E. coli, 16: A. baumannii, 17: P. aeruginosa and M: PCR Marker-100 bp ladder

Table 3: Details of the 17 representative isolates chosen for ERIC-PCR
CA: Carcinoma, DM: Diabetes mellitus, PVD: Peripheral vascular disease, IHD: Ischemic heart disease, COPD: Chronic obstructive pulmonary disease, UTI: Urinary tract infection, RTI: Respiratory tract infection, ET: Endotracheal and ND: Not documented, Cluster 1: Lane number 2 and 7, Cluster 2: Lane number 1, 8 and 9, Cluster 3: Lane number 11 and 16

These organisms frequently are found resistant to most antibiotics except colistin and, less consistently to tigecycline. Consequently, colistin and tigecycline (final resort antimicrobial agents) have been tried with limited success. The lack of effective antimicrobial agents demand newer agents for the treatment of infections caused by NDM-1-producing bacteria and other carbapenem resistant organisms (Menezes and Menezes, 2013). Rapid spread of CRE species in a hospital in Mumbai has been previously reported (Muir and Weinbren, 2010).

In our study, of the 53 blaNDM-1 positive cases, 08 (15%) had succumbed to death which was in accordance of report by Shenoy et al. (2014), in which 03 of the 61 (4.9%) patients had succumbed to death. Infections caused by NDM-1 producing pathogens are tough to treat leading to complications, but do not make pathogens more virulent or transmissible. Further, such infections range from mild to severe, while some have been fatal. The immuno-compromised status of the patient could be a predisposing factor for these infections (Menezes and Menezes, 2013).

Present study imparts insights into the intricate molecular epidemiology of blaNDM-1 gene in this tertiary care center. Among the different representative strains for which ERIC-PCR was performed, clonal relatedness was observed only with K. pneumoniae and A. baumannii isolates. Both the clonally related A. baumannii were isolated from endotracheal fluid. Clonal cluster type 1, consisting of 4 isolates of K. pneumoniae, were isolated from sputum, tissue, pleural fluid and urine. Clonal cluster type 2, consisting of 2 isolates of K. pneumoniae, were isolated from drain fluid from gall bladder and sputum. Though they were all isolated from different clinical samples, with the exception of A. baumannii, there is still a strong possibility of horizontal dissemination of blaNDM-1 gene within the healthcare setup. This calls for not only strict implementation of infection prevention and isolation protocols that could curtail possible outbreaks, but also routine surveillance of hospital environmental sampling and water supplies (Shanthi et al., 2013).

Clonal relatedness was however not observed in other isolates i.e., 3 isolates of P. aeruginosa and 3 isolates of E. coli. This is indicative of appearance of multiple clones with limited dissemination between patients, suggesting strong selection pressure on bacterial population, emphasizing the necessity for appropriate governance and administration of antibiotic therapy, within health-care units. Results demonstrate extensive diversity of blaNDM-1 producers which is consistent with the findings of previous studies (Shanthi et al., 2013; Kumarasamy et al., 2010; Nagaraj et al., 2012; Castanheira et al., 2011).

CONCLUSION

The blaNDM-1 gene has "An alarming potential" to spread and diversify among bacterial populations. This study demonstrated a high incidence of NDM-1-producing multi-drug resistant Gram negative bacilli from patients with different clinical diseases. There was clonal relatedness among K. pneumoniae and A. baumannii isolates and clonal diversity among P. aeruginosa and E. coli. This calls for increased alertness, continuous surveillance and strict enforcement of antibiotic policy with restricted use of inducer drugs. Carbapenem antibiotics should be treated as the last resort and reserved for severe infections. Spread of NDM-1 producing isolates seriously limit the options for clinical treatment. Thus, enhanced efforts are urgently needed to control the further spread of NDM-1-producing bacterial pathogens. It is very important to identify cases of NDM related infections early and prevent the spread by implementing screening, sanitation measures and isolation of the carriers. Hence, routine antimicrobial susceptibility testing along with genotypic characterization should be performed.

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