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Environmental Microbiology (EM) | Microbial Ecology and Diversity
Microbiol. Biotechnol. Lett. 2023; 51(3): 268-279
Taslin Jahan Mou1,2†, Nasrin Akter Nupur1†, Farhana Haque1, Md Fokhrul Islam2,3, Md. Shahedur Rahman4, Md. Amdadul Huq5, and Anowar Khasru Parvez1*
1Department of Microbiology, Jahangirnagar University, Savar-1342 Dhaka, Bangladesh
2Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, The University of Edinburgh, United Kingdom-EH9 3FF
3Department of Pharmacy, Jahangirnagar University, Savar-1342 Dhaka, Bangladesh
4Department of Genetic Engineering and Biotechnology, Jashore University of Science and Technology, Jashore-7408, Bangladesh
5Department of Food and Nutrition, College of Biotechnology and Natural Resource, Chung-Ang University, Anseong 17546, Republic of Korea
Correspondence to :
Anowar Khasru Parvez, email@example.com
The pharmaceutical industry in Bangladesh produces a diverse range of antibiotics for human and animal use, however, waste disposal management is inadequate. This results in substantial quantities of antibiotics being discharged into water bodies, which provide suitable environment for the growth of antibiotic-resistant bacteria, capable of spreading resistance genes. This study intended for exploring the bacterial antibiotic resistance profile in adjoining aquatic environmental sources of pharmaceutical manufacturing facilities in Bangladesh. Seven surface water samples were collected from the vicinity of two pharmaceutical industries located in the Savar area and 51 Escherichia coli isolates were identified using both phenotypic and genotypic methods. Antibiotic susceptibility tests revealed the highest percentage of resistance against ampicillin, azithromycin, and nalidixic acid (100%) and the lowest resistance against meropenem (1.96%) out of sixteen different antibiotics tested. 100% of the study E. coli isolates were observed with Multidrug resistance phenotypes, with the Multiple Antibiotic Resistance (MAR) value ranging from 0.6-1.0. Furthermore, 69% of the isolates were Extended Spectrum Beta-Lactamases (ESBL) positive as per the Double Disk Diffusion Synergy Test (DDST). ESBL resistance genes blaTEM, blaCTX-M-13, blaCTX-M-15, and blaSHV were detected in 70.6% (n = 36), 60.8% (n = 32), 54.9% (n = 28), and 1.96% (n = 1) of the isolates, respectively, by Polymerase Chain Reaction (PCR). Additionally, 15.68% (n = 8) of the isolates were positive for E. coli specific virulence genes in PCR. These findings suggest that pharmaceutical wastewater, if not properly treated, could be a formidable source of antibiotic resistance spread in the surrounding aquatic environment. Therefore, continued surveillance for drug resistance among bacterial populations around drug manufacturing facilities in Bangladesh is necessary, along with proper waste disposal management.
Keywords: Surface water sample, pharmaceutical industries, multi drug resistance, Escherichia coli, ESBL resistance genes, virulence genes
Antibiotic pollution in the environment is a growing global concern due to its adverse impact on human, animal and environmental health . Prolonged exposure of environmental microbes to antibiotics nurture the development and transmission of antibiotic resistance , posing a significant challenge to medical practice and public health . Therefore, from the One Health perspective, it is urgent to uncover the role of environment in the evolution and spread of antibiotic resistance . Anthropogenic activities, including those associated with medical facilities, pharmaceutical industries, municipalities, agriculture, and aquaculture, contribute to the transmission of antibiotic-resistant bacteria and their determinants in the environment [4, 5].
Pharmaceutical waste and wastewater contaminated with antibiotics and other compounds has become a topic of concern due to their potential to impose selective pressure on the microbiota in the environment, even at low concentrations [6, 7]. A study reported high levels of antibiotics from multiple groups in pharmaceutical wastewater and receiving waters in South Asian countries, used for both human and animal infection . Frequent use and misuse of antibiotics, inadequate wastewater treatment system and meteorological conditions are some of the factors contributing to the occurrence of antibiotic pollution in these regions.
The pharmaceutical industry in Bangladesh primarily focuses on drug formulation and manufacturing of finished products. As per the Bangladesh Association of Pharmaceutical Industries (BAPI) and Directorate General of Drug Administration (DGDA), there are currently 257 licensed pharmaceutical companies in the country. Unfortunately, the waste disposal and wastewater treatment practices in Bangladesh are inadequate . As a result, the water bodies surrounding drug manufacturing facilities receive untreated wastewater, leading to a plethora of drug resistant bacteria and related genes in the environment . However, still there is no study reported on the presence of antibioticresistant and ESBL-producing
In the current study, seven surface water samples were collected from different locations near and around two pharmaceutical industries located in the Savar area of Bangladesh, which is a hub for the pharmaceutical industry. Sample collection period was June 2020 to December 2020. Sterile 500 ml Schott Duran's bottles (Germany) were used to collect the samples, sealed tightly and transported immediately to the laboratory in an insulated ice box. Temperature and pH of the samples were measured using a mercury thermometer graduated from 0℃ to 100℃ and a glass electrode pH meter (SCHOTT instrument), respectively.
The bacteriological quality of the collected water samples was assessed using standard conventional culture methods. Briefly, using sterile normal saline (0.85%) the samples were serially diluted up to 10-4 and 100 μl from each dilution was inoculated onto nonselective nutrient agar (NA) (Oxoid, UK) for the total bacterial count enumeration. To determine the total gram-negative bacteria, selective MacConkey agar (HIMEDIA) was used. Additionally, ESBL-producing bacteria were counted using ESBL Chrom-agar supplemented with ceftriaxone antibiotic (stock solution prepared at 0.57 mg/ml), where the incubation was at 37℃ for 24 h. The growth of individual colonies on each plate was recorded and enumerated.
The presumptively identified
Two sets of primer UAL-754 and UAR-900 were used to amplify the
Antibiotic sensitivity of the isolates was evaluated by the standard Kirby-Bauer disk diffusion method employing Mueller-Hinton agar medium (Oxoid Limited, England), in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (2020) . The study utilized 16 antibiotic disks from eight different groups-Ampicillin (AM), Amoxycillin+Clavunic acid (AMC), Azithromycin (AZM), Amikacin (AK), Gentamycin (GN), Chloramphenicol (C), Cefixime (CFM), Cefuroxime (CXM), Ceftriaxone (CRO), Ceftazidime (CAZ), Cefotaxime (CTX), Ciprofloxacin (CIP), Levofloxacin (LEV), Nalidixic acid (NA), Meropenem (MEM), and Tetracycline (TE). These antibiotics are frequently used and produced in Bangladesh.
To test for ESBL production, the Double Disk Diffusion Synergy Test (DDST) was performed. The test inoculum was spread onto Mueller-Hinton agar (MHA) after matching the turbidity to 0.5 McFarland. An augmenting disk (20 μg amoxicillin + 10 μg clavulanic acid) was placed on the surface of MHA, and then disks of ceftriaxone (30 μg), ceftazidime (30 μg), and cefotaxime (30 μg) were positioned around it, ensuring that each disk was 15− 20 mm away from the augmenting disk (center to center). The spacing between the disks was adjusted as needed for each strain to accurately detect synergy.
Each bacterial isolate was subjected to characterization of their ESBL-genotypes and the presence of virulence factors. Specific regions of the
Multiplex polymerase chain reaction (PCR) was conducted using four primer sets to detect the virulence genes
Bacterial isolates of this study were identified by 16S rRNA PCR using universal primers 8F (5-AGT TTG ATC CTG GCT CAG-3) and 1492R (5-ACC TTG TTA CGA CTT-3), following a previously established protocol . Among the 16S rRNA-positive isolates, seven representative isolates (chosen randomly from each collected sample) were selected for further analysis of the 16S rRNA gene sequence. Besides the representative isolates, one isolate positive for three ESBL genes and high MAR index was chosen for the sequencing as well. Big-Dye Terminator cycle sequencing kit and an ABI Prism 310 Genetic Analyzer (Applied Biosystems Inc., USA) were employed to determine the amplicon nucleotide sequences. Chromas 2.6.5 (Technelysium, Australia) was used to analyze the Chromatograms of the sequences followed by identification by BLAST search. Multiple sequence alignment was performed using the ClustalW Multiple Alignment algorithm in BioEdit 7.2.6 software and submitted to the NCBI for accession numbers. Phylogenetic relationship of 16S rRNA gene sequences were analyzed using the partial sequence of these amplicons and reference sequences with MEGA 11. Neighbor-joining method was employed to build the phylogenetic tree using the bootstrap replicates (1000).
Temperature of the water samples were in the range from 28.9℃ to 30.5℃, whereas pH values were observed from 6.3 to 7.8. The total viable bacterial count of the samples varied from 5.0 × 105 cfu/ml to >TNTC, with total
100% of the study
Out of the total 51
The results of 16S rRNA analysis indicate that the majority of the samples are closely related to a strain of
Antibiotic resistance have emerged as a serious threat for global public health, as it hinders the efficacy of treatment which results in increased mortality, morbidity, and associated healthcare costs. Aquatic environments connected to industrial wastewater and municipality sewage system become highly polluted with antibiotics and resistant bacteria containing mobile genetic determinants [23, 24]. Wastewaters discharged into environment without any prior or proper treatment promote the dissemination of antibiotic resistance and their genetic determinants in the inhabitant bacterial community [25, 26]. Drug manufacturing facilities has been recognized as a major hotspot for spreading antibiotic resistance as per comparative review analysis on possible source of the resistance from environmental health perspective .
Here, the temperature of the samples were in the range of 26.4℃ to 30℃, while the pH values were differed from 6.3 to 7.8 (slightly alkaline). According to the Department of Environment (DoE) standard in Bangladesh, the parameters were within the permissible limit for domestic, irrigation utilization and others [29, 30]. The presence of bacterial community and their distribution could be significantly influenced by the temperature of the water habitat . Moreover, bacterial susceptibility to antibiotic stress is dependent on the abiotic factors like temperature and pH, with having difference among the pathogenic and non-pathogenic strains . In a previous study pathogenic strains of
Total bacterial count in the water samples were 5 × 105 cfu/ml to TNTC, while the gram-negative bacterial count ranged from TFTC to 8.1 × 105 cfu/ml. This observation is consistent with previous studies on surface water samples, indicating a high population density of bacteria in environmental water samples affected by human and industrial activities . This concur the findings of a study conducted in Borneo that observed higher viable bacterial counts with values from 1.6 × 104 to 3.0 × 104 cfu/ml in groundwater influenced by human or industrial activities, as compared to the counts in water samples distant from such activities . The presumptive count of ESBL isolates ranged from TFTC to 5.1 × 105 cfu/ml, suggesting a high abundance of ESBL-producing bacteria in the water samples. The findings are somewhat similar to other studies, which observed predominance of ESBL bacteria in the aquatic environment and wastewater discharge from various sources [35, 36].
Herein our study, 100% (n = 51) of the isolates showed multidrug resistance against eight different group of antibiotics (Table 2). An earlier study reported similarly 100% MDR
Multiple Antibiotic Resistance (MAR) value was greater than > 0.2 for all of the study
We found 69% (n = 35) of
Four different ESBL genes were targeted in this study to be detected from the
The wastewater treatment system in Bangladesh is inadequate, resulting in the discharge of poorly treated or untreated effluents that contain a significant number of active antimicrobial agents and antibiotic-resistant bacteria (ARB). Our study provides valuable insight into the extent of antibiotic pollution and distribution of ARB in aquatic environmental sources surrounding pharmaceutical industries in Bangladesh. High MAR index values observed in this study indicate the high risk of antibiotic pollution of the sampling sources, underscoring the urgent need for proper legislation and regulation of waste disposal from industrial activities. Finally, continuous and systematic monitoring of the drugresistant pattern of the commensal bacterial community is necessary for effective policy formulation and implementation.
The investigation has been supported by grants from the University Grant Commission (UGC), Ministry of Science and Technology, Bangladesh and Jahangirnagar University.
Conceptualization: Taslin Jahan Mou, Anowar Khasru Parvez
Data curation: Nasrin Akter Nupur, Farhana Haque
Formal analysis: Nasrin Akter Nupur, Taslin Jahan Mou, Md Fokhrul Islam, Md. Shahedur Rahman
Funding acquisition: Taslin Jahan Mou, Anowar Khasru Parvez, Fokhrul Islam
Investigation: Nasrin Akter Nupur, Taslin Jahan Mou
Methodology: Taslin Jahan Mou, Anowar Khasru Parvez, Md. Shahedur Rahman
Project administration: Taslin Jahan Mou, Anowar Khasru Parvez
Resources: Md. Shahedur Rahman, Md. Amdadul Huq
Software: Md. Shahedur Rahman
Supervision: Taslin Jahan Mou, Anowar Khasru Parvez
Validation: Md. Shahedur Rahman, Md. Amdadul Huq
The authors have no financial conflicts of interest to declare.
Wongwarut Boonyanugomol , Worrarat Kongkasem , Prasit Palittapongarnpim , Myunghwan Jung , Min-Kyoung Shin , Hyung-Lyun Kang , Seung-Chul Baik , Woo-Kon Lee and Myung-Je ChoMicrobiol. Biotechnol. Lett. 2018; 46(3): 261-268 https://doi.org/10.4014/mbl.1802.02002