|Year : 2020 | Volume
| Issue : 1 | Page : 23-27
The Presence of gram-negative bacteria carrying the New Delhi metallo-β-lactamase gene on abiotic touch surfaces at a tertiary care center
Archana Agrawal, Chakrakodi N Varun, Anjali Shette, Daisy Vanitha John, Ravikumar Raju
Department of Neuromicrobiology, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India
|Date of Submission||11-Oct-2019|
|Date of Decision||23-Jan-2020|
|Date of Acceptance||28-Mar-2020|
|Date of Web Publication||12-Jun-2020|
Dr. Ravikumar Raju
Professor, Department of Neuromicrobiology, National Institute of Mental Health and Neurosciences, Hosur Road, Wilson Garden, Bengaluru - 560 029, Karnataka
Source of Support: None, Conflict of Interest: None
Objectives: New Delhi metallo-β-lactamase (NDM), which has emerged as a major mechanism of resistance to carbapenems in Gram-negative bacteria (GNB), challenges effective patient management of health-care systems. Nonpathogenic environmental bacteria present on abiotic touch surfaces in hospitals may serve as reservoirs for the NDM gene and contribute to the emergence and spread of resistance to carbapenems. The aim of this study was to determine the presence of NDM-positive GNB in the environment of a tertiary care center. Materials and Methods: Fifty-eight swab samples were collected from various touch surfaces in 12 different wards between January and February 2017. The swabs were cultured in nutrient broth and subsequently subcultured onto McConkey agar plates. Both lactose and nonlactose fermenting colonies grown were identified by biochemical methods. The polymerase chain reaction method was used to detect NDM carriage. Results: Twenty-seven (46%) of the samples were positive for microbial growth, of which 21 (36%) samples yielded bacterial growth on McConkey agar plates. Of the 30 isolates identified, 25 (83%) were nonfermenting GNB (NFGNB) and 5 (17%) were Klebsiella spp., of which 1 was Klebsiella oxytoca. NFGNB were isolated mostly from tables and infusion stands in various wards. Four of the five Klebsiella spp. were from patients' beds. Two isolates of NFGNB and one Klebsiella spp. were positive for the NDM gene. Conclusion: In addition to serving as potential pathogens of nosocomial infections, environmental bacteria present on abiotic touch surfaces in hospitals may serve as reservoirs for the NDM gene.
Keywords: Hospital environment, Klebsiella, New Delhi metallo-β-lactamase, nonfermenting Gram-negative bacteria, touch surfaces
|How to cite this article:|
Agrawal A, Varun CN, Shette A, John DV, Raju R. The Presence of gram-negative bacteria carrying the New Delhi metallo-β-lactamase gene on abiotic touch surfaces at a tertiary care center. Biomed Res J 2020;7:23-7
|How to cite this URL:|
Agrawal A, Varun CN, Shette A, John DV, Raju R. The Presence of gram-negative bacteria carrying the New Delhi metallo-β-lactamase gene on abiotic touch surfaces at a tertiary care center. Biomed Res J [serial online] 2020 [cited 2020 Aug 14];7:23-7. Available from: http://www.brjnmims.org/text.asp?2020/7/1/23/286560
| Introduction|| |
Carbapenems are beta-lactam antibiotics used as last-line agents in critically ill patients for treating a variety of bacterial infections. The emergence of resistance to carbapenems has been reported in multidrug-resistant Gram-negative bacteria (MDRGNB) that are often associated with nosocomial infections. MDRGNB-related infections cause high morbidity and mortality in hospitalized patients due to a lack of therapeutic options. New Delhi metallo-β-lactamase (NDM), a carbapenemase enzyme that hydrolyzes beta-lactam antibiotics, has emerged as one of the major mechanisms of resistance to carbapenems in Gram-negative bacteria (GNB). NDM was initially detected in a multidrug-resistant Klebsiella pneumoniae isolate from a Swedish patient with a urinary tract infection hospitalized in India. Clinical isolates of Escherichia More Details coli-producing NDM were found in Indian health-care facilities as early as 2006 (2006). Based on epidemiological links, the Indian subcontinent is considered as the main reservoir of NDM producers., The presence of NDM-positive isolates in patients with no history of foreign travel or any association with India suggests that the bacteria can also be acquired through local transmission.
The gene product of NDM (blaNDM) is a 27.5-kDa protein of 269 amino acids. Twenty-one variants of NDM gene have been reported based on differences in one or two amino acid residues at different positions. Sequencing-based studies show that the NDM gene was commonly found on large conjugative plasmids in members of the Enterobacteriaceae family, whereas it was located on plasmids or chromosomes in Acinetobacter and Pseudomonas. The insertion sequence ISAba125 serves as a promoter for this gene and aids mobility between different bacterial genera. NDM-positive GNB are widely disseminated in hospitals. Outbreaks of NDM-positive GNB have been reported in intensive care units (ICUs), mostly in patients at extreme ages, with fatal outcomes in many cases.,
Various evidence suggests that antibiotic resistance genes in human pathogens have environmental origins., Nonpathogenic environmental bacteria present on touch surfaces in hospitals may serve as reservoirs for the NDM gene and contribute to the emergence and spread of antibiotic resistance by horizontal gene transfer (HGT) through mobile genetic elements. Some environmental bacteria are resistant to heat, ultraviolet light, and chemical sanitizers, making disinfection by routine methods ineffective. Their persistence on abiotic touch surfaces and their ability to form biofilms on medical devices increase the risk of transmission through the hands of health-care workers, resulting in colonization and subsequent infection in patients.,, We previously reported NDM-positive clinical isolates of MDRGNB in patients admitted to different wards of the hospital. The present study was undertaken to determine the presence of GNB-carrying NDM gene on the environmental surfaces of various wards.
| Materials And Methods|| |
From January to February 2017, 58 environmental samples were collected from 12 different locations, including various wards (neurology [n = 8], neurosurgery [n = 6], head injury [n = 7], stroke [n = 6], operation theaters [OT] [OT1 to OT4, 5 each], the emergency ICU [n = 6], and verandas [n = 5]). A sterile swab, dipped in nutrient broth, was used to collect samples from 10 cm2 area from patients' beds, bedside tables, infusion stands, trolleys, treatment tables, ventilators, microscopes, walls, and floors. The samples were transported to the laboratory for microbiological and molecular assays.
The swabs were incubated in 5 ml of nutrient broth at 37°C for 24 h. Tubes showing turbidity were cultured on McConkey agar and subcultured further to obtain pure cultures. Both lactose fermenting and nonlactose fermenting bacteria were subjected to Gram staining and conventional biochemical method of identification. All isolates were then stored at −80°C in Luria–Bertani broth, supplemented with 20% glycerol for molecular assays.
Detection of the New Delhi metallo-β-lactamase gene by polymerase chain reaction
Total genomic DNA was extracted from the isolates by the alkaline lysis method. A polymerase chain reaction (PCR) assay for the NDM gene was performed with specific primers (NDM-F 5′-GGGCAGTCGCTTCCAACGGT-3′ and NDM-R 5'-GTAGTGCTCAGTGTCGGCAT-3'). The 475-bp internal fragment was amplified in a 25-μl reaction mixture using a Veriti 96-well thermal cycler (Applied Biosystems, Life Technologies, Foster City, CA, USA). The thermal cycling conditions used were as follows: initial denaturation for 5 min at 94°C, 30 cycles of 30 s at 95°C for denaturation, 30 s at 60°C for annealing, extension for 30 s at 72°C, and a final extension at 72°C for 5 min. Gel electrophoresis was done using a 1.5% agarose gel, and the amplified PCR products were visualized using a gel documentation system (Bio-Rad laboratories, Mumbai, India). The K. pneumoniae ATCC BAA-2146 strain was used as a positive control for the NDM gene.
| Results|| |
Of the 58 environmental samples cultured, 27 (46%) samples were positive for microbial growth. None of the samples collected from the OTs (OT1 to OT4) or verandas showed microbial growth. A direct smear of the positive cultures showed both Gram-positive cocci and GNB in 23 (40%) samples, GNB in 2 (3%) samples, and fungi in 2 (3%) samples. Culture on McConkey agar showed bacterial growth in 21 (36%) samples, of which 12 (20%) samples contained a single type of bacterium and 9 (15%) samples contained two types of bacteria. Subculture of mixed cultures was done on McConkey agar plates to isolate pure cultures. Of 30 bacterial isolates obtained, 25 (83%) were nonlactose fermenters and 5 (17%) were lactose fermenters, with mucoid colonies. All the nonlactose fermenters were identified as nonfermenting GNB (NFGNB) by biochemical tests, as they were negative for indole, mannitol, and triple sugar iron test. They were isolated from swabs collected from patients' beds (n = 4), bedside tables (n = 5), infusion stands (n = 5), treatment tables (n = 5), trolleys (n = 3), walls (n = 2), and curtains (n = 1) in the pediatric, neurology, neurosurgery, head injury and stroke wards, and emergency ICU [Table 1]. Of the five lactose fermenters, all were Klebsiella spp., of which one was Klebsiella oxytoca. Four of these isolates were cultured from patients' beds, and one was cultured from a treatment table. The PCR assay revealed the presence of the NDM gene in two NFGNB cultured from a treatment table and walls of the neurosurgery and stroke wards and in one Klebsiella spp. cultured from a patient's bed in the emergency ICU. The significance of these isolates in nosocomial infections was not established.
|Table 1: Details of culture-positive environmental samples collected from the hospital environment|
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| Discussion|| |
NDM-positive MDRGNB are a major cause of concern in nosocomial-associated infections in health-care settings. Prolonged survival of bacteria in the environment and HGT may contribute to an increase in the incidence of these MDRGNB., In our previous study, screening by direct PCR showed the presence of the NDM gene in 49% of the environmental swab samples collected from various touch surfaces in different wards of the hospital. In the present study, we isolated and identified NDM-positive bacteria from environmental samples. All samples collected from female neurosurgery and stroke wards showed bacterial growth. There was no difference in the isolation rate from various surfaces in different wards. None of the samples collected from the OTs (OT1 to OT4) showed bacterial growth, indicating good infection control measurements practiced in these facilities.
NFGNB (25/30, 83%) and Klebsiella spp. (5/30, 17%) were the only GNB isolated from environmental samples collected from the various wards in this study. Most of the NFGNB were isolated from tables and infusion stands, whereas four of the five Klebsiella spp. were from patients' beds. Isolation of Acinetobacter baumannii from emergency ICU ventilators and bed rails was reported in China, indicating that equipment and touch surfaces in emergency ICUs may have bacterial colonization. In the present study, of the 30 GNB isolated from the environmental samples, one Klebsiella spp. and two NFGNB were positive for the NDM gene, as shown by the PCR. The higher NDM positivity (49%) obtained by direct PCR in our earlier study as compared with the present study (10%) could be due to amplification of the NDM gene from nonviable bacteria or bacterial species that are not supported by McConkey agar. Previous research demonstrated that NDM-positive K. pneumoniae survived for up to 1 month on stainless steel surfaces and that the transfer of the NDM gene from Klebsiella spp. to other bacteria occurred with increasing numbers over time at room temperature. NDM-producing A. baumannii was isolated from sewage of hospitals in Beijing, China, but not from river and drinking or fishpond water, suggesting that HGT between organisms can occur in sewage. The authors concluded that NDM-associated hospital-acquired infections may be caused directly by A. baumannii or bacterial genera that acquired the resistance gene from A. baumannii. NDM-positive K. pneumoniae from wash basins and A. baumannii and Stenotrophomonas maltophilia from infants' body sites were isolated at a sick newborn care unit (SNCU) in a rural hospital in West Bengal, India. The authors reported that imipenem-resistant E. coli that caused septicemia and imipenem-susceptible E. coli isolated from infants' body sites were clonally identical in this facility. They speculated that the NDM gene might have been disseminated in the SNCU via various GNB and that this gene was subsequently transferred to imipenem-susceptible E. coli isolates that caused septicemia in newborns.
The finding of NDM-positive NFGNB on a treatment table and Klebsiella spp. in the emergency ICU in this study is important, as they may cause fatal infections in immunocompromised patients with comorbidities. We previously reported isolation of NDM-positive multidrug-resistant A. baumannii and K. pneumoniae causing respiratory and urinary tract infections, respectively, in patients admitted to various wards. Thus far, we have not performed antibiotic susceptibility or molecular typing for NDM-positive environmental isolates of GNB. Others reported no evolutionary relationship between clinical and environmental isolates of A. baumannii based on antibiotic and pulsed field gel electrophoresis profiles.
NFGNB present on environmental surfaces probably serve as a reservoir for the NDM plasmid, as previous reports show that this gene emerged in Acinetobacter spp. prior to its dissemination among Enterobacteriaceae. In Acinetobacter, the NDM gene is part of a Tn125 composite transposon, bracketed between two copies of the ISAba125 element, which was originally identified in NDM-negative A. baumannii environmental isolates. Sequence analysis of NDM-carrying plasmid shows that the plasmid backbone has ultraviolet light resistance genes (mucAB), which gives the bacteria a survival advantage in the environment. Plasmids carrying the NDM gene carried other genes that conferred resistance to several antibiotic classes and those affecting virulence and pathogenesis. Resistance to carbapenems, together with the ability of these pathogens to form biofilms on inserted devices, such as urinary catheters, may contribute to colonization of these isolates in patients.
Human factors, such as travel to countries with a high incidence of NDM producers, uncontrolled or inappropriate use of antibiotics, overcrowding, and limited laboratory diagnosis and infection-control strategies, have led to the global spread of NDM gene. Bacterial-related factors, including environmental persistence, prolific HGT, and hospital-adapted clones, allow bacteria to serve as reservoirs of NDM and contribute to the local spread of this gene. Treatment options for NDM producers are very limited, although they are susceptible to polymyxins (colistin), glycylcyclines (tigecyclines), and fosamycin. Resistance to polymyxins and tigecyclines has been observed in recent years, and there are no prospects of newer drugs on the market at this time. The dissemination of the NDM gene among GNB demands infection-control measures to prevent the spread of GNB-carrying NDM gene in health-care facilities.
| Conclusion|| |
We found three GNB isolates collected from the hospital environment harboring NDM gene. In addition to serving as potential pathogens of nosocomial infections, environmental bacteria present on abiotic touch surfaces in hospitals may serve as reservoirs for the NDM gene.
Financial support and sponsorship
Archana Agarwal and Chakrakodi N. Varun are recipients of Senior Research Fellowship from the University Grants Commission, Government of India. Daisy Vanitha John is supported by the Women Scientist Scheme of Department of Science and Technology, Government of India.
Conflicts of interest
There are no conflicts of interest.
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