This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on http://www.researchprotocols.org, as well as this copyright and license information must be included.
Antimicrobial resistance is a widespread, alarming issue in global health and a significant contributor to human death and illness, especially in low and middle-income countries like Bangladesh. Despite extensive work conducted in environmental settings, there is a scarcity of knowledge about the presence of resistant organisms in the air.
The objective of this protocol is to quantify and characterize the airborne resistomes in Bangladesh, which will be a guide to identify high-risk environments for multidrug-resistant pathogens with their spatiotemporal diversity.
This is a cross-sectional study with an environmental, systematic, and grid sampling strategy focused on collecting air samples from different outdoor environments during the dry and wet seasons. The four environmental compartments are the frequent human exposure sites in both urban and rural settings: urban residential areas (n=20), live bird markets (n=20), rural households (n=20), and poultry farms (n=20). We obtained air samples from 80 locations in two seasons by using an active microbial air sampler. From each location, five air samples were collected in different media to yield the total bacterial count of 3rd generation cephalosporin (3GC) resistant
The study started in January 2018, and the collection of air samples was completed in November 2018. We have received 800 air samples from 80 study locations in both dry and wet seasons. Currently, the laboratory analysis is ongoing, and we expect to receive the preliminary results by October 2019. We will publish the complete result as soon as we clean and analyze the data and draft the manuscript.
The existence of resistant bacteria in the air like those producing extended-spectrum beta-lactamases, carbapenem-resistant
DERR1-10.2196/14574
Antimicrobial resistance is considered a rapidly progressive global public health issue with the potential of environmental transmission to a larger extent. However, very little information is available on the transmission of antimicrobial resistance through the air. Additionally, the capacity to carry and propagate resistance of these resistomes is poorly studied worldwide. There is a recognized need to examine the existence of such bacteria that have the ability to confer resistance through atmospheric air. Bangladesh is an important location to study this pathway. This study can provide critical insight into antimicrobial resistance transmission and help determine where efforts could be implemented to reduce environmental transmission.
Antimicrobial resistance is a widespread and alarming issue in global health, causing more than 700,000 deaths every year [
Based on few studies conducted on the presence of antimicrobial resistance in the environment, the spatial and seasonal diversity of antibiotic resistant bacteria are well established [
Additionally, several anthropogenic events also enhance the ability of antibiotic resistance genes to be transferred horizontally and pose a further risk for the environment to act as a reservoir for resistant bacteria [
The main study objective is to detect the existence of resistomes in the air samples from outdoor environments of Bangladesh, which carry genes that confer resistance to antibiotics with temporal and spatial diversity. We hypothesize that the outdoor environment (air) in Bangladesh acts as a reservoir for bacteria carrying genes that confer resistance to antibiotics with temporal and spatial diversity. The specific objectives of the study are as follows:
To determine the prevalence of antibiotic-resistant bacteria in the air samples of outdoor environments (both poultry and residential) in Bangladesh that carry genes conferring resistance to antibiotics.
To characterize antibiotic-resistant organisms using different phenotypic and genotypic methods.
To explore the clonal relationship between antimicrobial resistant organisms isolated from high- and low-risk areas.
To identify the temporal and spatial distribution of resistant bacteria in outdoor environments of Bangladesh.
This is a cross-sectional pilot study designed to collect air samples from high-risk and low-risk environments in urban and periurban settings of Bangladesh, mainly the Dhaka metropolitan area and Mirzapur Upazilla (subdistrict) of Tangail District. Environmental systematic and grid sampling will be followed according to approaches described by Keith [
Sampling strategy and framework.
Mapping with GIS software (ArcGIS, Esri, Redlands, California) will plot concentrations of resistant genes and antimicrobial-resistant bacteria in environmental compartments (high- and low-risk areas) in each location using GPS coordinates. Temporal variation will be observed by comparing the magnitude of resistant bacteria and genes in the dry season with those in the wet season.
Air samples from commercial poultry farms, live bird markets, rural households, and urban residential areas will be collected. All active sampling will be performed using the same Surface Air System Sampler (SAS Super 180 Microbial Air Sampler, Bioscience International, Rockville;
Active microbial air sampler.
According to the manufacturers’ instruction and using QIAamp DNA Mini Kit (QIAGEN, Germany), total DNA from the culture sweeps will be extracted after counting colonies on SPC agar plates. The colonies will then be incubated for 44 hours at 37°C. High-throughput sequencing will be performed using an Illumina MiSeq sequencing system (Illumina, San Diego, CA). The downstream analysis quality will be ensured by removing raw reads, with an average quality score below 20 or with length less than 100 bp (101 bp in length) or having three or more ambiguous nucleotides. The high-throughput sequencing will be carried out by the “Index 101 PE” (paired-end sequencing; 101-bp reads and 8-bp index sequence) sequencing strategy. There will be almost the same quantity of clean reads in this manner for each sample. The processing of the call sequences as well as raw fluorescent images will be carried out through a base-calling pipeline (Sequencing Control Software, Illumina). Metagenomic analyses will be carried out with the filtered clean reads (almost 1.6 GB per sample) after removal of the raw reads contaminated by an adapter (>15 bp overlap) or having three or more “N” [
All the culture plates (except SPC) exposed to air during air sampling will be incubated at 37°C, with 44 hours for SB agar and 18-24 hours for MacConkey agar and MSA. After incubation, each plate will be counted for both types of colonies (typical and atypical). To achieve clean/pure cultures, 3-4 phenotypically different isolated colonies will be subcultured from each plate on corresponding antibiotic-supplemented agar media. Following incubation (mentioned earlier), culture sweeps from each plate will be dissolved in tryptic soy broth cryovials containing 30% glycerol. These will be preserved at –80°C for future usage.
By using different antibiotic discs, the agar diffusion test will determine the antimicrobial susceptibility as per the Clinical and Laboratory Standards Institute (CLSI 2016) guidelines [
Polymerase chain reaction for genes specific for each of the resistance phenotypes (for ESBL: CTX-M, TEM, SHV; for carbapenem: NDM; for VRE: vanA; for MRSA: mecA) will be carried out according to the procedures described previously [
Phenotypically same isolates (with or without similar resistance pattern) obtained from different locations or time periods will be further tested by pulsed-filed gel electrophoresis to determine their clonal diversity and dispersion.
Plasmid DNA extraction and analysis from resistant isolates will be executed through the rapid alkaline lysis method and horizontal gel electrophoresis in 0.8% agarose gels, respectively [
The primary objective of this study is to determine the prevalence of antibiotic resistant organisms with resistance gene characterization in air samples from different locations and during different seasons. However, there are no baseline data available about measuring antimicrobial resistance in air samples. No prior assumptions were possible to show the anticipated variation in different exposures or seasonal contexts, although some prior knowledge has been utilized [
The outcome variables that will be assessed are as follows:
The prevalence of antibiotic-resistant organisms and resistance genes (positive occurrence of resistant bacteria/genes as a proportion of the number of samples) from each environmental location
The geospatial distribution of antibiotic resistant organisms and antibiotic resistance genes in study areas
The temporal prevalence of resistant bacteria and concentration of resistant genes in dry and wet season to assess seasonal variation in antimicrobial resistance
The identification of high-risk environments for air borne pollution with antimicrobial resistance
In this study, we will determine the presence of resistant organisms in air samples by total counting (colony-forming units per liter) at different study sites in Bangladesh. Significant differences in carriage rates of antibiotic-resistant organisms (and concentrations of resistant genes) in high- and low-risk environments will be determined by Chi-square and independent
The Research Review Committee and Ethical Review Committee of International Centre for Diarrhoeal Disease Research, Bangladesh, have approved this research protocol (protocol number: PR-17048). A unique study identification number was assigned to all air samples to ensure anonymity of the study sites. The study started in January 2018, and the collection of air samples was completed in November 2018. We have received 800 air samples from 80 study locations in both dry and wet seasons. Currently, the laboratory analysis is ongoing, and we expect to receive the microbiological results by October 2019. After completion of data cleaning and analysis, the manuscript submission is anticipated to be submitted before fall 2020. In addition to publication in a high-impact, peer-reviewed journal and as per the dissemination plan, the study results will be shared with the study participants, with scientific communities, with relevant government authorities, and in related conferences or workshops.
The rise and spread of superbugs have become a key public health and planetary health concern worldwide. Infections caused by multidrug-resistant bacteria are linked to greater mortality rates than antimicrobial-susceptible bacteria [
The burden of the antimicrobial resistance rate depends on the population of a country and its environment. Bangladesh is a highly populated country; therefore, the rate of antimicrobial resistance is extensive due to rapid spread of antimicrobial-resistant organisms. Antimicrobial resistance genes are conferring their resistance value to a wider community including both animals and humans, through close interactions with the environment and the wastes that are disposed in the environment, directly affecting the food chain [
Intrinsic genetic determinants of resistance factors are harbored by bacteria with macromolecules in the environment. Robust evidence suggests that such macromolecules developing “environmental resistomes” are a source from which clinically relevant bacteria acquire antibiotic resistance genes [
The importance of environmental compartments as the transmission hub of antimicrobial resistance is well established. However, airborne resistomes and their transmission pathway are poorly studied. Without containment of environmental reservoirs, antimicrobial resistance prevention policy will fail [
Regarding the strengths, our study will focus on the air resistomes, which is a less explored environmental dimension of antimicrobial resistance transmission dynamics. To our knowledge, this is the first study to explore the presence of superbugs in the air in commonly exposed areas in Bangladesh. The limitation exists in the small sample size and lack of baseline data from the atmospheric environment of Bangladesh. Therefore, the findings may not be generalizable for all areas in the country.
Peer-reviewer report from the International Centre for Diarrhoeal Disease Research, Bangladesh.
Basic Local Alignment Search Tool
Carbapenem-resistant
Clinical and Laboratory Standards Institute
Extended-Spectrum Beta-Lactamase
Methicillin-resistant
Vancomycin-resistant
This research protocol was funded by The Swedish International Development Cooperation Agency (SIDA; contribution no. 54100089). The International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) acknowledges with gratitude the commitment of SIDA to its research efforts. The icddr,b is also grateful to the Governments of Bangladesh, Canada, Sweden, and the UK for providing core/unrestricted support. MA is supported by National Institutes of Health Fogarty International Centre Global Health Equity Scholars program (D43TW010540).
MA and MAI conceptualized the idea and developed the study protocol and field methods with study supervision; MA, MAI, MIH, SRS, and NA formulated the laboratory protocols; MA, MAI, and MRI worked on the sample size estimation and analysis plan; MIH and SRS are responsible for the sample processing and laboratory experiments; MA wrote the original draft; and all authors reviewed, edited, and contributed substantially to writing the manuscript.
None declared.