Abstract
The diversity of antibiotic-resistance bacteria (ARB) from the effluents of Aizawl city municipal waste dumping site was studied using a culture-dependent method. The present study molecularly identified 73 isolates that were differentiated into three phyla (Proteobacteria, Actinobacteria, and Firmicutes) belonging to 17 family and 22 genera. Bacillus constitutes the most dominant genus comprising 16% of the total isolates. The topology of the phylogenetic tree differentiates them into five major clades. Corynebacterium and Rhodococcus which are morphologically alike were clustered together and the Gram-positive bacteria such as Staphylococcus, Bacillus, and Lysinibacillus formed a separate cluster. Antibiotic resistance of the identified bacterial isolates was performed using 19 different antibiotics. Among the isolates, 70 of them found resistant to polymixin B and nalidixic acid and 10 isolates exhibited resistance to 15 tested antibiotics. The present study revealed that bacteria with antibiotic resistance are extensively distributed in the effluents of the dumping site and may serve as a significant reservoir for the spreading of antibiotic resistance to opportunistic pathogens.
Electronic supplementary material
The online version of this article (10.1007/s13205-020-02548-z) contains supplementary material, which is available to authorized users.
Keyword: ARB, Args, MDR, Opportunistic pathogens, Dumping site
Introduction
One of the globally concern with regards to human and animal health is the emergence of antimicrobial resistance, the spread of antibiotic-resistant bacteria (ARB), and the associated antibiotic-resistant genes (ARGs) (World Health Organization 2014). This evolution of multidrug-resistant (MDR) pathogens led to severe problems for the clinicians in treating the infectious diseases due to the reduced pharmaceutical potential of available drugs. (Zhang et al. 2009; Bello-López et al. 2019).
Effluents of dumping sites is considered as the hotspot for antimicrobial resistance, accumulating a cocktail of several antibiotic compounds generally from human wastes and excrement, which possibly contain multidrug-resistant pathogens (Gao et al. 2012). Although information of pathogenic bacteria in the natural environment is limited, effluents provide a suitable environment for the exchange of ARGs among the MDR bacteria and other environmental bacteria through horizontal gene transfer, which further acts as a vehicle for the transmission of various disease-causing pathogens (Holmes et al. 2016; Bello-López et al. 2019). Upon reaching the water bodies such as rivers, the effluent can easily transmit pathogenic bacteria between humans and animals that results in broader epidemiological issues (Galvin et al. 2010).
Understanding the bacterial population in natural environments provides direct evidence of opportunistic pathogens and possible health risks associated and help to make appropriate strategies to prevent diseases (Ibekwe et al. 2013). Detection and identification of pathogenic bacteria using polymerase chain reaction (PCR)-based 16S rRNA gene amplification and sequencing is the most common, rapid, and reliable method (Albinana-Gimenez et al. 2009). Thus, an in-depth microbiological investigation will reveal a broad understanding of such environments (Mwaikono et al. 2016).
The municipal solid waste consists of solids, semi-solids, or liquids, mainly from houses, commercial or industrial premises (Nyangababo and Hamya 1986). An increase in population and urbanization increases the amount and variety of wastes worldwide, which may pose serious health and environmental risk if not managed properly (Guerrero et al. 2013). Moreover, inadequate resources and lack of proper management systems create dumping sites that contaminate the river ecosystems through its effluents leading to the spread of human pathogenic bacteria and ARGs in both terrestrial and aquatic ecosystems (Blomström et al. 2016).
Open dumping of municipal solid wastes remains the predominant waste disposal option in developing countries. Mizoram is a northeast state of India and Aizawl is the capital densely populated city. The local administration department uses the municipal solid waste dumpsite as a solid waste dumpsite for Aizawl city since 2nd May 1997. According to Aizawl Municipal, Dept. of Sanitation wing, more than 106.2 tons of garbage is dumped every day. Below this dumpsite, two streams called Damte and Damdiai (local names) falls which finally joins in one of the most important river of Mizoram called Tuirial River. No such specific study has been carried out to assess the bacterial populations present in these contaminated sites. Consequently, it has become essential to monitor the micro-flora associated with these contaminated sites. Therefore, the present study aims to identify the bacterial communities, their diversity, and abundance at a selected waste dumping site for pathogenic/ARB based on their antibiotic-resistance patterns.
Methods
Sample collection
Sampling was carried out from the effluents of municipal solid waste dumpsite in the eastern part of Aizawl city around 30 km from the city during March 2015. The dump-site sediment samples were collected in a sterile screw-capped tube and brought into Molecular Microbiology and Systematics Laboratory, Department of Biotechnology, Mizoram University. The samples were stored at 4 ℃ until it was processed for isolation.
Isolation of culturable bacteria
Isolation of bacteria was carried out using the serial dilution method and spread plate technique, as described by Kuster and Williams (1964). Four different nutritional media, namely starch casein agar (SCA), glycerol asparagine agar (ISP5), Tyrosine agar medium (ISP7), King’s modified medium, and LB medium, were used for the isolation of bacteria. Serial dilution was performed by preparing a stock solution of 1 gm of sample in 9 ml of sterile distilled water in a test tube and agitated for 10 min. The suspension was serially diluted up to dilutions of 10–3 and spread on different media. It was then incubated at 28 ± 2 °C, and the pure colonies of the bacteria isolates were purified by subsequent sub-culturing in their respective growth media (Fig. 1).
Fig. 1.
Genus wise abundance of the bacteria isolated from dumping site represented in percentage (%); the color indicates different Genera of the isolated bacterial isolates
Molecular characterization of bacterial isolates
Genomic DNA was isolated using a bacterial DNA extraction kit (Invitrogen Life Technologies) as per the manufacturer’s protocol. Quantification of genomic DNA was performed using 0.8% agarose gel electrophoresis using a Bio-rad Gel Doc XR + system. Applied biosystems thermal cycler PCR (Veriti™) was used for the amplification of 16S rRNA gene fragment using universal primers PA: 5′-AGAGTTTGATCCTGGCTCAG-3′) and PH: 5′-AAGGAGGTGATCCAGCCGCA-3′ (Qin et al. 2009). PCR reactions and conditions were executed following our previous paper (Zothanpuia et al. 2016). The amplified products were purified using Pure-link PCR Purification Kit (Invitrogen Life Technologies) and were sequenced at State level biotech hub, Department of Biotechnology, Mizoram University. The obtained 16S rRNA gene sequences were deposited in the National Centre for Biotechnology Information (NCBI) GenBank database, and accession numbers were obtained. These sequences were then compared for similarity percentage with the reference strains of bacteria from the NCBI genomic database, using Basic Local Alignment Search Tool (BLASTn) search available at http://www.ncbi.nlm.nih.gov/ blast website. Type strains with the highest similarity percentage were retrieved from the EzTaxon database, and multiple sequence alignment was performed using Clustal-W software available at http://www.ebi.ac.uk/clustalw (Thompson et al. 1997). Phylogenetic analysis was performed using MEGA 6.0 software using the maximum-likelihood method and Tamura–Nei parameters algorithm (Tamura et al. 2013). The significance of the branching order was determined by bootstrap analysis of 1000 alternative trees. Trees were viewed and edited using program Fig Tree 1.3.1.
Antibiotic sensitivity profiling
Purified bacteria isolates were grown in nutrient broth and incubated at 28 ± 2 °C, 150 rpm for 24 h. The grown bacterial suspensions were spread on Muller Hinton agar (MHA) medium using a sterile L-shaped spreader. The plates were dried before placing the diffusion discs containing antibiotics and were incubated at 28 ± 2 °C for 24 h. The isolates susceptibility to 19 different antibiotics was performed following the Kirby Bauer disk diffusion method as described by Robert et al. (2009) as per National Committee for Clinical Laboratory Standards (NCCLS).
Nineteen (n = 19) standard commercially available disks containing antibiotics such as Methillicin (Met5), Kanamycin (K5), Ampicillin (Amp10), Streptomycin (S10), Erythromycin (E15), Norfloxacin (NX10), Sparfloxacin (SPX5), Tetracycline (TE30), Gentamicin (Gen10), Chloramphenicol (C10), Polymixin B (PB50), Ciprofloxacin (CIP10), Vancomycin (VA10), Trimethoprim (TR10), Clindamycin (CD10), Levofloxacin (LE5), Nalidixic acid (NA30), Cefixime (CFM5), and Azithromycin (AZM15) were used. Concentrations of each tested antibiotic are mentioned in superscript in microgram (µg) as per the EUCAST (2019) (European Committee on Antimicrobial Susceptibility Testing) standards. Antibiotic sensitivity was observed by measuring the inhibition zone’s diameters in mm and categorized as resistant, intermediate, and sensitive to antibiotics (Passari et al. 2015).
Results
Isolation of culturable bacteria
In the present investigation, more than 250 colonies of bacteria were observed and 73 microbial cultures were purified and isolated from the effluents of Aizawl municipal solid waste dumping site based on their varying characters and morphological patterns on varying solid media (Fig. S1, Table S1). All 73 microbes were subsequently identified based on their 16S rRNA gene amplification (Fig. S2). Identified isolates divides into three phyla (Proteobacteria, Actinobacteria, and Firmicutes), 17 family, and 22 genera. All the gene sequences were deposited in the NCBI GenBank database, and accession numbers obtained were MG711554–MG711583, MH793369–MH793399, and MH793400–MH793411, respectively (Table 1). Among the isolates, 60% (n = 44) are Gram-positive bacteria while 40% (n = 30) belongs to Gram-negative group. Bacillus constitutes the most dominant genus comprising 16% of the total isolates, followed by Alcaligenes, Arthrobacter, Achromobacter, Lysinibacillus, Staphylococcus, Corynebacterium, Brachybacterium, Brevibacterium, Nocardiopsis, Rhodococcus, Leucobacter, Streptomyces, Enterococcus, Aneurinibacillus, Pseudomonas, Stenotrophomonas, Brevundimonas, Kocuria, Micrococcus, Tsukamurella and Zhihengliuella.
Table 1.
Taxonomic description of the bacterial isolates obtained from the effluent of dumping site
| S. no | Isolate name and strain no | Closest relative species in BLAST and accession no | % Similarity | GenBank accession number |
|---|---|---|---|---|
| 1 | Brevibacterium sp. DST143 |
Brevibacterium sp. (KM507608) |
99% | MG711554 |
| 2 | Rhodococcus sp. DST144 |
Rhodococcus sp. (FN357284) |
99% | MG711555 |
| 3 | Nocardiopsis sp. DST 145 |
Nocardiopsis sp. (KU382690) |
99% | MG711556 |
| 4 | Arthrobacter sp. DST 146 |
Arthrobacter sp. (KR906432) |
99% | MG711557 |
| 5 | Leucobacter sp. DST 147 |
Leucobacter sp. (KX289462) |
99% | MG711558 |
| 6 | Streptomyces sp. DST 148 |
Streptomyces sp. (KP99845) |
99% | MG711559 |
| 7 | Brachybacterium sp. DST 149 |
Brachybacterium sp. (FJ357630) |
99% | MG711560 |
| 8 | Nocardiopsis sp. DST150 |
Nocardiopsis sp. (JN942114) |
97% | MG711561 |
| 9 | Arthrobacter sp. DST151 |
Arthrobacter sp. (KM362724) |
99% | MG711562 |
| 10 | Corynebacterium sp. DST152 |
Corynebacterium sp. (CP009251) |
97% | MG711563 |
| 11 | Brevibacterium sp. DST153 |
Brevibacterium sp. (AM398220) |
99% | MG711564 |
| 12 | Leucobacter sp. DST154 |
Leucobacter sp. (KJ461710) |
99% | MG711565 |
| 13 | Corynebacterium sp. DST155 |
Corynebacterium sp. (KF428998) |
99% | MG711566 |
| 14 | Brachybacterium sp. DST156 |
Brachybacterium sp. (KF875581) |
100% | MG711567 |
| 15 | Tsukamurella sp. DST157 |
Tsukamurella sp. (JN695025) |
99% | MG711568 |
| 16 | Arthrobacter sp. DST158 |
Arthrobacter sp. (KJ619485) |
97% | MG711569 |
| 17 | Arthrobacter sp. DST159 |
Arthrobacter sp. (KJ642536) |
97% | MG711570 |
| 18 | Corynebacterium sp. DST160 |
Corynebacterium sp. (KF428998) |
100% | MG711571 |
| 19 | Arthrobacter sp. DST161 |
Arthrobacter sp. (KT724297) |
99% | MG711572 |
| 20 | Arthrobacter sp. DST162 |
Arthrobacter sp. (KJ619485) |
99% | MG711573 |
| 21 | Arthrobacter sp. DST163 |
Arthrobacter sp. (KR906430) |
99% | MG711574 |
| 21 | Arthrobacter sp. DST164 |
Arthrobacter sp. (KM035405) |
99% | MG711575 |
| 23 | Brachybacterium sp. DST165 |
Brachybacterium sp. (KP027820) |
98% | MG711576 |
| 24 | Kocuria sp. DST166 |
Kocuria sp. (GU318369) |
99% | MG711577 |
| 25 | Brachybacterium sp. DST167 |
Brachybacterium sp. (DQ399745) |
99% | MG711578 |
| 26 | Corynebacterium sp. DST168 |
Corynebacterium sp. (KF428998) |
100% | MG711579 |
| 27 | Brevibacterium sp. DST 169 |
Brevibacterium sp. (DQ347560) |
98% | MG711580 |
| 28 | Micrococcus sp. DST 170 |
Micrococcus sp. (KJ685828) |
97% | MG711581 |
| 29 | Zhihengliuella sp. DST 171 |
Zhihengliuella sp. (JF274915) |
99% | MG711582 |
| 30 | Rhodococcus sp. DST 172 |
Rhodococcus sp. (GU143683) |
99% | MG711583 |
| 31 | Staphylococcus sp. DST173 |
Staphylococcus sp. (MK015791) |
99% | MH793369 |
| 32 | Bacillus sp. DST174 |
Bacillus sp. (KY379939) |
99% | MH793370 |
| 33 | Bacillus paralicheniformis DST175 |
Bacillus paralicheniformis (MN013960) |
99% | MH793371 |
| 34 | Alcaligenes faecalis DST176 |
Alcaligenes faecalis (MN176578) |
99% | MH793372 |
| 35 |
Aneurinibacillus aneurinilyticus DST177 |
Aneurinibacillus aneurinilyticus (HQ238588) |
99% | MH793373 |
| 36 | Bacillus cereus DST178 |
Bacillus cereus (MG021182) |
99% | MH793374 |
| 37 | Pseudomonas sp. DST179 |
Pseudomonas sp. (JQ660558) |
99% | MH793375 |
| 38 | Alcaligenes sp. DST180 |
Alcaligenes sp. (HG316114) |
99% | MH793376 |
| 39 | Alcaligenes faecalis DST181 |
Alcaligenes faecalis (MN176578) |
99.34% | MH793377 |
| 40 | Lysinibacillus sp. DST182 |
Lysinibacillus sp. (MH260942) |
99.04% | MH793378 |
| 41 | Lysinibacillus sp. DST183 |
Lysinibacillus sp. (MK757955) |
99.85% | MH793379 |
| 42 | Bacillus cereus DST184 |
Bacillus cereus (MG561362) |
99% | MH793380 |
| 43 | Staphylococcus sp. DST185 |
Staphylococcus sp. (LC190797) |
99% | MH793381 |
| 44 | Achromobacter sp. DST186 |
Achromobacter sp. (KX302621) |
99.88% | MH793382 |
| 45 | Achromobacter sp. DST187 |
Achromobacter sp. (LC487320) |
99.58% | MH793383 |
| 46 | Alcaligenes faecalis DST188 |
Alcaligenes faecalis (KX344024) |
99.07% | MH793384 |
| 47 | Enterococcus sp. DST189 |
Enterococcus sp. (FJ514022) |
98.78% | MH793385 |
| 48 | Lysinibacillus sp. DST190 |
Lysinibacillus sp. (MN199553) |
99.88% | MH793386 |
| 49 | Alcaligenes faecalis DST191 |
Alcaligenes faecalis (MN176578) |
99.71% | MH793387 |
| 50 | Bacillus sp. DST192 |
Bacillus sp. (HM567060) |
98.75% | MH793388 |
| 51 | Bacillus cereus DST193 |
Bacillus cereus (MH010181) |
99.51% | MH793389 |
| 52 | Alcaligenes sp. DST194 |
Alcaligenes sp. (KC478943) |
99.76% | MH793390 |
| 53 | Stenotrophomonas sp. DST195 |
Stenotrophomonas sp. (MH465188) |
99.50% | MH793391 |
| 54 | Bacillus sp. DST196 |
Bacillus sp. (HM567060) |
99.75% | MH793392 |
| 55 | Brevundimonas diminuta DST197 | Brevundimonas diminuta (KU685402) | 99.72% | MH793393 |
| 56 | Lysinibacillus sp. DST198 |
Lysinibacillus sp. (MN199553) |
97% | MH793394 |
| 57 | Lysinibacillus sp. DST199 |
Lysinibacillus sp. (MN199553) |
100% | MH793395 |
| 58 | Achromobacter sp. DST200 |
Achromobacter sp. (LC487320) |
99.80% | MH793396 |
| 59 | Staphylococcus lentus DST201 |
Staphylococcus lentus (KT261262) |
99.90% | MH793397 |
| 60 |
Achromobacter xylosoxidans DST202 |
Achromobacter xylosoxidans (MN061281) |
100% | MH793398 |
| 61 | Bacillus sp. DST203 |
Bacillus sp. (KU851838) |
99.48% | MH793399 |
| 62 | Bacillus sp. DST204 |
Bacillus sp (MN187270) |
98.82% | MH793400 |
| 63 | Achromobacter sp. DST205 |
Achromobacter sp. (LC487320) |
98.84% | MH793401 |
| 64 | Alcaligenes faecalis DST206 |
Alcaligenes faecalis (MH793409.) |
99.81% | MH793402 |
| 65 | Alcaligenes faecalis DST207 |
Alcaligenes faecalis (KR827435) |
100% | MH793403 |
| 66 | Achromobacter sp. DST208 |
Achromobacter sp. (KP974274) |
99.34% | MH793404 |
| 67 | Bacillus sp. DST209 |
Bacillus sp. (KJ849835) |
99.55% | MH793405 |
| 68 | Alcaligenes faecalis DST210 |
Alcaligenes faecalis (KR827435.) |
99.89% | MH793406 |
| 69 | Bacillus sp. DST211 |
Bacillus sp. (MG917734) |
99.75% | MH793407 |
| 70 | Bacillus thuringiensis DST212 |
Bacillus thuringiensis (KY362202) |
100% | MH793408 |
| 71 | Alcaligenes faecalis DST213 |
Alcaligenes faecalis (KX118704) |
99.20% | MH793409 |
| 72 | Alcaligenes faecalis DST214 |
Alcaligenes faecalis (KX462790) |
98.60% | MH793410 |
| 73 | Staphylococcus sciuri DST215 |
Staphylococcus sciuri (JX134627) |
99.71% | MH793411 |
Phylogenetic analysis
The phylogenetic tree was constructed taking all identified isolates along with their type-strains retrieved from the EzTaxon database using Maximum-likelihood (ML) and Tamura Nei (TN93 + G) parameter with lowest Bayesian Information Criterion (BIC) values (22482) and highest Akaike Information Criterion (AICs) value (20275) (Fig. 2). An estimated value of transition/transversion bias (R) is 1.03. The topology of the tree generated differentiated the isolates into five major clades. Corynebacterium and Rhodococcus which are morphologically alike were clustered together, other actinobacteria such as Streptomyces and Nocardiopsis were clustered together. The Gram-positive bacteria such as Staphylococcus, Bacillus, and Lysinibacillus also formed a separate clade.
Fig. 2.
Maximum-likelihood (ML) phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic relationship between the isolates. Numbers at branches indicate bootstrap values in 1000 replicates.: indicate bacterial isolates obtained from the effluents of the dumping site
Antibiotic-resistance bacteria
All the isolates identified from the effluent of the dumping site were screened for their antibiotic sensitivity. Among them, ten isolates (Brevibacterium sp. DST143, Arthrobacter sp. DST161, Brachybacterium sp. DST156, Achromobacter sp. DST205, Achromobacter sp. DST187, Achromobacter sp. DST208, Lysinibacillus sp. DST182, Brevundimonas diminuta DST197, Bacillus thuringiensis DST212, and Achromobacter xylosoxidans DST202) showed resistance to more than 15 antibiotics out of 19 antibiotics tested (Table S2). Brevibacterium sp. DST143 and Arthrobacter sp. DST161 showed resistant to 18 antibiotics, while the Brachybacterium sp. DST156, Achromobacter sp. DST205, and Achromobacter sp. DST187 showed resistance to 16 antibiotics. From a total of 73 isolates studied for their antibiotic sensitivity, 70 isolates showed resistance to polymixin B and Nalidixic acid whereas all the isolates were found susceptible to levofloxacin [Fig. S3 (A), Fig. S3 (B), Table S2].
Discussion
Open dumping sites outside the cities are a major source of pathogenic and antibiotic-resistance microbes that can transit to human. According to the Aizawl Municipal Department of Sanitation wing, approx. 106 tons/day of garbage are dumped every day. There is no segregation of waste, so all the wastes are simply dumped in this region. Many of these wastes enter the stream (directly or indirectly) and the effluents that reached the aquatic ecosystems without pretreatment make the water quality unfit for human uses. The study site is attached to a small stream that later joins an important river, a source of livings for the local people. The effluents harboring the bacteria which could possibly reach the river and can be a major threat for the spread of ARB (Cheng et al. 2014; Kraemer et al. 2019), and consumption of the water contaminated with such microbes can also pose risk to public health (Pandey et al. 2014). There are certain bacteria renowned as etiological agents for several waterborne diseases and epidemics. Therefore, understanding the bacterial population in those dumping sites is of primary importance in knowing the gene flow of antimicrobial resistance.
The present study revealed the abundance and resistance pattern of bacteria from the effluents of Aizawl municipal solid waste dumping site and identified them. Among the diverse bacteria obtained, few genera included such as Staphylococcus, Pseudomonas, Corynebacterium, and Micrococcus, which were reported earlier as opportunistic pathogens being able to cause infections during its proximity or contact to skin, mucous membrane, nasopharyngeal cavity, respiratory ducts, and urogenital passages (Tamminen et al. 2011). A high degree of antibiotic resistance was observed in actinobacteria such as Brevibacterium and Corynebacterium isolated from the river Godavari that could enhance the waterborne infections (Jani et al., 2019) and was in accordance with the present study. Although, no systematic investigation is been carried out to detect the antibiotic-resistant bacteria from Aizawl municipal solid waste dumping sites, however, it was reported that antibiotic-resistant microbes existed within nearby localities (Hauhnar et al. 2018). It is reported that Gram-negative bacteria Morganella morgana found to be highly resistant to chloramphenicol and ciprofloxacin isolated from Aizawl civil hospital wastewater. Moreover, Bacillus cereus showed high resistance towards ampicillin and kanamycin which was isolated from State Referral Hospital wastewater (Hauhnar et al. 2018). The present study also reported the isolation of Bacillus cereus resistant to seven antibiotics. This municipal solid wastewater is an important vehicle for the transport of antimicrobial-resistance bacteria and associated genes. Infections by staphylococci has been one of the most common within the state of Mizoram (Lalhmingmawii et al. 2018) where the present investigation also reported the isolation of several Staphylococcus resistant to more than ten antibiotics.
There are several reports on the occurrence and proliferation of pathogenic antibiotic-resistance bacteria and ARGs in several water systems such as urban rivers infesting the water bodies and its vicinity (Iwane et al. 2001; Hu et al. 2008; Watkinson et al. 2007; Servais and Passerat 2009). There was also a report on the increased levels of antibiotic resistance in an urban stream of Jiulongjiang River, China (Ouyang et al. 2015) which also supported the present investigation. Injudicious use of antimicrobials, inadequate treatment of waste waters, and improper discard of livestock animals were important drivers of AMR reported recently (Taneja and Sharma 2019). The ARGs related to the bacterial pollutants can multiply in the hosts, transfer to other bacterial inhabitants and be subject to additional development and progression in the bacterial community (Alonso et al. 2001). As such ARGs that occur in the environment can potentially cause risks to human health and the sanctity of the environment (Andy and Okpo 2018). Therefore, there is a necessity for physical, chemical and microbiological evaluation of water quality which needs to be recorded and information to be delivered on the safety of the water to safeguard sustained protection of water source to the communities.
Conclusions
The present investigation determined the prevalence of antibiotic-resistant bacteria that included opportunistic pathogens belonging to the genera of Pseudomonas, Staphylococcus, Bacillus, Corynebacterium, and Nocardiopsis from the effluents of the municipal dumping site in Aizawl, India. Given this fact, proper treatment and segregation of the wastes are very much necessary to avoid the outbreak of antibiotic-resistant pathogens. Moreover, it is a huge task to functionally characterize environmental microbiomes by conventional isolation method and culturing them; there are many unculturable microbes which need to be taken care when public health is considered. Moreover, with the advances in high-throughput sequencing and computational biology, the investigation of microbial communities based on culture-independent approaches using metagenomics, can further be of great help in the study in characterizing diverse pathogenic and antibiotic-resistant bacteria.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgments
The authors are thankful to the Department of Biotechnology, Government of India, New Delhi for financial support under DBT’s Unit of Excellence program for NE (102/IFD/SAN/4290-4291/2016-2017). The senior author is thankful to NIFTEM authorities for their general support while compiling the manuscript.
Author contributions
Z and BPS: planned the project, arranged the funds, designed the experiments, and wrote the manuscript. Z, RZ and VVL: conducted the survey, collected isolates, cultured, did DNA isolation and antibiotic sensitivity profiling study. Z, VVL, AKP and MKY: conducted the phylogenetic profiling, sequence depositions, prepared tables and figures and wrote the discussion.
Compliance with ethical standards
Conflict of interest
All authors read and approved the final manuscript for publication and with no conflict of interests among the authors.
References
- Albinana-Gimenez N, Clemente-Casares P, Calgua B, Huguet JM, Courtois S, Girones R. Comparison of methods for concentrating human adenoviruses, polyomavirus JC and noroviruses in source waters and drinking water using quantitative PCR. J Virol Methods. 2009;158:104–109. doi: 10.1016/j.jviromet.2009.02.004. [DOI] [PubMed] [Google Scholar]
- Alonso A, Sanchez P, Martínez JL. Environmental selection of antibiotic resistance genes. Environ Microbiol. 2001;3(1):1–9. doi: 10.1046/j.1462-2920.2001.00161.x. [DOI] [PubMed] [Google Scholar]
- Andy IE, Okpo EA. Occurrence and antibiogram of bacteria isolated from effluent and waste dump site soil of selected hospitals in Calabar metropolis, Nigeria. Microbiol Res J Int. 2018;25(5):1–9. doi: 10.9734/MRJI/2018/44932. [DOI] [Google Scholar]
- Bello-López JM, Cabrero-Martínez OA, Ibáñez-Cervantes G, Hernández-Cortez C, Pelcastre-Rodríguez LI, Gonzalez-Avila LU, Castro-Escarpulli G. Horizontal gene transfer and its association with antibiotic resistance in the Genus Aeromonas spp. Microorganisms. 2019;7(9):363. doi: 10.3390/microorganisms7090363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blomström AL, Lalander C, Komakech AJ, Vinnerås B, Boqvist S. A metagenomic analysis displays the diverse microbial community of a vermicomposting system in Uganda. Infect Ecol Epidemiol. 2016;6:32453. doi: 10.3402/iee.v6.32453. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng D, Liu X, Wang L, Gong W, Liu G, Fu W, Cheng M. Seasonal variation and sediment-water exchange of antibiotics in a shallower large lake in North China. Sci Total Environ. 2014;476–477:266–275. doi: 10.1016/j.scitotenv.2014.01.010. [DOI] [PubMed] [Google Scholar]
- EUCAST: European Committee on Antimicrobial Susceptibility Testing, breakpoint tables for interpretation of MIC’s & zone diameters. (2019). version 9.0, valid from 01.01.2019. Available from: http://www.eucast.org/fileadmin/src/media/PDFs/EUC
- Galvin S, Boyle F, Hickey P, Vellinga A, Morris D, Cormican M. Enumeration and characterization of antimicrobial-resistant Escherichia coli bacteria in effluent from municipal, hospital, and secondary treatment facility sources. Appl Environ Microbiol. 2010;76(14):4772–4779. doi: 10.1128/AEM.02898-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gao P, Munir M, Xagoraraki I. Correlation of tetracycline and sulfonamide antibiotics with corresponding resistance genes and resistant bacteria in a conventional municipal wastewater treatment plant. Sci Total Environ. 2012;421–422:173–183. doi: 10.1016/j.scitotenv.2012.01.061. [DOI] [PubMed] [Google Scholar]
- Guerrero LA, Maas G, Hogland W. Solid waste management challenges for cities in developing countries. Waste Manag. 2013;33(1):220–232. doi: 10.1016/j.wasman.2012.09.008. [DOI] [PubMed] [Google Scholar]
- Hauhnar L, Pachuau L, Lalhruaitluanga H. Isolation and characterization of multi-drug resistant bacteria from hospital wastewater sites around the city of Aizawl, Mizoram. Adv Biosci Biotechnol. 2018;9(7):311–321. doi: 10.4236/abb.2018.97020. [DOI] [Google Scholar]
- Holmes AH, Moore LS, Sundsfjord A, Steinbakk M, Regmi S, Karkey A, Guerin PJ, Piddock LJ. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet. 2016;387(10014):176–187. doi: 10.1016/S0140-6736(15)00473-0. [DOI] [PubMed] [Google Scholar]
- Hu J, Zacharek S, He YJ, Lee H, Shumway S, Duronio RJ, Xiong Y. WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1-CUL4-ROC1 ligase. Genes Dev. 2008;22(7):866–871. doi: 10.1101/gad.1624008. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ibekwe AM, Leddy M, Murinda SE. Potential human pathogenic bacteria in a mixed urban watershed as revealed by pyrosequencing. PLoS ONE. 2013;8(11):e79490. doi: 10.1371/journal.pone.0079490. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Iwane T, Urase T, Yamamoto K. Possible impact of treated wastewater discharge on incidence of antibiotic resistant bacteria in river water. Water Sci Technol. 2001;43(2):91–99. doi: 10.2166/wst.2001.0077. [DOI] [PubMed] [Google Scholar]
- Jani K, Bandal J, Rale V, Shouche Y, Sharma A. Antimicrobial resistance pattern of microorganisms isolated and identified from Godavari River across the mass gathering event. J Biosci. 2019;44:121. doi: 10.1007/s12038-019-9941-z. [DOI] [PubMed] [Google Scholar]
- Kraemer SA, Ramachandran A, Perron GG. Antibiotic pollution in the environment: from microbial ecology to public policy. Microorganisms. 2019;7(6):180. doi: 10.3390/microorganisms7060180. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuster E, Williams ST. Selection of media for isolation of Streptomycetes. Nature. 1964;202:928–929. doi: 10.1038/202928a0. [DOI] [PubMed] [Google Scholar]
- Lalhmingmawii L, Laldinmawii G, Lalremruati J, Lalzirtitri A, Malvi Y. Speciation and antibiogram of staphylococcal isolates from a tertiary care hospital of North East India. Int J Med Sci Clin Invent. 2018;5(10):4086–4088. [Google Scholar]
- Mwaikono KS, Maina S, Sebastian A, Schilling M, Kapur V, Gwaksia P. High-throughput sequencing of 16S rRNA gene reveals substantial bacterial diversity on the municipal dumpsite. BMC Microbiol. 2016;16:145. doi: 10.1186/s12866-016-0758-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nyangababo JT, Hamya JW. The deposition of Lead, Cadmium, Zinc and Copper from motor traffic on Bradaria enimi and soil along a major Bombo road in Kampala city. Int J Environ Stud. 1986;27(1–2):115–119. doi: 10.1080/00207238608710280. [DOI] [Google Scholar]
- Ouyang WY, Huang FY, Zhao Y, Li H, Su JQ. Increased levels of antibiotic resistance in urban stream of Jiulongjiang River, China. Appl Microbiol Biotechnol. 2015;99(13):5697–5707. doi: 10.1007/s00253-015-6416-5. [DOI] [PubMed] [Google Scholar]
- Pandey PK, Kass PH, Soupir ML, Biswas S, Singh VP. Contamination of water resources by pathogenic bacteria. AMB Express. 2014;4(1):51. doi: 10.1186/s13568-014-0051-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Passari AK, Mishra VK, Saikia R, Gupta VK, Singh BP. Isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential. Front Microbiol. 2015;6:273. doi: 10.3389/fmicb.2015.00273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Qin S, Li J, Chen HH, Zhao GZ, Zhu WY, Jiang CL, Xu LH, Li WJ. Isolation, diversity and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rain forests in Xishuangbanna, China. Appl Environ Microbiol. 2009;75:6176–6186. doi: 10.1128/AEM.01034-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robert AP, Lorraine F, Walter M, Ronald MR. Laboratory exercises in microbiology. 3. New Jersey, United States: John Wiley and Sons, Inc. Hoboken; 2009. pp. 1–288. [Google Scholar]
- Servais P, Passerat J. Antimicrobial resistance of fecal bacteria in waters of the Seine river watershed (France) Sci Total Environ. 2009;408:365–372. doi: 10.1016/j.scitotenv.2009.09.042. [DOI] [PubMed] [Google Scholar]
- Tamminen M, Karkman A, Lõhmus A, Muziasari WI, Takasu H, Wada S, Suzuki S, Virta M. Tetracycline resistance genes persist at aquaculture farms in the absence of selection pressure. Environ Sci Technol. 2011;45(2):386–391. doi: 10.1021/es102725n. [DOI] [PubMed] [Google Scholar]
- Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biology Evol. 2013;30(12):2725–2729. doi: 10.1093/molbev/mst197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taneja N, Sharma M. Antimicrobial resistance in the environment: the Indian scenario. Indian J Med Res. 2019;149(2):119–128. doi: 10.4103/ijmr.IJMR_331_18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25(24):4876–4882. doi: 10.1093/nar/25.24.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watkinson AJ, Murby EJ, Costanzo SD. Removal of antibiotics in conventional and advanced wastewater treatment: implications for environmental discharge and wastewater recycling. Water Res. 2007;41(18):4164–4176. doi: 10.1016/j.watres.2007.04.005. [DOI] [PubMed] [Google Scholar]
- Zhang Y, Marrs CF, Simon C, Xi C. Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp. Sci Total Environ. 2009;407(12):3702–3706. doi: 10.1016/j.scitotenv.2009.02.013. [DOI] [PubMed] [Google Scholar]
- Zothanpuia PAK, Gupta VK, Singh BP. Detection of antibiotic-resistant bacteria endowed with antimicrobial activity from a freshwater lake and their phylogenetic affiliation. Peer J. 2016;4:e2103. doi: 10.7717/peerj.2103. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.