Introduction

Riverine ecosystems are under significant pressure from heavy metal and antibiotic contamination, resulting in domination of the native species by the resistant and tolerant species; thus, causing an alteration of the bacterial community structure [1,2,3]. Heavy metal and antibiotic-tolerant bacteria isolated from polluted river systems across the globe have demonstrated tolerance to copper (Cu), lead (Pb), nickel (Ni), cobalt (Co), chromium (Cr), cadmium (Cd), zinc (Zn), and mercury (Hg). In addition, reports show a high incidence of multiple antibiotic-resistant (MAR) bacteria with resistance to cephalosporins, quinolones, sulphonamides, and aminoglycosides [4, 5]. Survival strategies evolved by bacteria to overcome metal toxicity vary from metal sequestration (extracellular or intracellular) to active mechanisms (metal-specific efflux or uptake repression), and enzymatic detoxification (metallic ion reduction and/or volatilization) [6].

Metal tolerance towards a wide range of divalent cations like Co2+, Zn2+, Cd2+, Cu2+, Fe2+, and Ni2+ have arisen from metal ion-specific efflux complexes belonging to resistance-nodulation-cell division (RND), cation diffusion facilitator (CDF) and PIB-type ATPase superfamilies [7]. The proton-driven antiporters or RND proteins facilitate metal ions efflux across the membrane and aid metal resistance in Gram-negative bacteria [8]. While, CDF proteins use the microbial Zn2+, but Co2+, Ni2+, Cd2+, and Fe2+ as primary substrates to energize removal of metal ions from the cytoplasm [7]. The PIB-type ATPases are ATP-coupled pumps driving the export and import of metal ions in bacteria [7, 9, 10].

Genetic adaptations like the target bypass (dfr and sul genes), efflux pumps (tet and cml genes), antibiotic inactivation (bla and str genes), and target modification (erm, mecA, and van genes) allow bacteria to develop resistance against antibiotics [11]. The location of many recognized antibiotic resistance genes in transposons, integrons, or plasmids have permitted gene mobilization and transfer to other bacteria [12]. In addition, the processes of co-resistance and cross resistance allowed bacteria to employ mechanisms that are common to both heavy metal and antibiotic resistance. Consequently, there is a very real and growing concern that metal stress can promote antibiotic resistance [13]. Multidrug resistance efflux pumps are typical examples of determinants conferring cross resistance to different antimicrobials. Beyond the conventional antimicrobials, the efflux pumps actively extrude a variety of compounds such as heavy metals, dyes, detergents, and organic compounds [14].

The resistance conferred by the superfamily RND efflux pumps play a vital role in the innate bacterial resistance and most prevalent in Gram-negative bacteria. There are four such efflux systems recognized in Pseudomonas aeruginosa namely, MexAB-OprM [15], MexCD-OprJ [16], MexEF-OprN [17] and MexXY-OprM [18]. Mex B, MexD, MexF, and MexY are the chemiosmotic efflux pumps while MexA, MexC, MexE and MexX belong to membrane fusion proteins, and OprM, OprJ and OprN are outer membrane channel-forming proteins. SdeAB [19] of Serratia and AdeABC and AdeDE [20] in Acinetobacter provide resistance to several antibiotics in a similar manner. These efflux pumps are part of the intrinsic resistance mechanism of bacteria towards tetracyclines, quinolones, chloramphenicol, macrolides, beta-lactams, and novobiocin [21]. Antimicrobial resistance (AMR) engendered by the presence of antibiotic resistance genes (ARG), is a characteristic feature of microorganisms. Antimicrobial resistance is evident even in samples taken from pristine environments, albeit at lower frequencies than in samples from human impacted environments [22]. Consequently, the unchecked discharge of antimicrobials in rivers can render surface waters hotspots of the resistant bacterial phenotypes by accelerated co-selection. Thus, rivers are being explored as potential sources of bacteria with antimicrobial resistant genes. The presence of Pseudomonas and Serratia in riverine ecosystems with multidrug resistance traits have been reported from various studies [23,24,25]. These isolates contain multidrug efflux pumps conferring multidrug resistance in mobile genetic elements like plasmids, integrons and transposons [26]. Moreover, horizontal gene transfer (HGT) is a basic feature prevalent in prokaryotes, engaged in transfer of resistance (antibiotic and heavy metal) and virulence genes in bacteria [27]. In this study, anthropogenic impact on the Teesta River was assessed via the spread of antibiotic and metal tolerance genes in specific isolate species; and substantiated by HGT between them.

Material and Methods

Determination of Minimum Inhibitory Concentration of the Heavy Metals Towards Bacterial Isolates

Species of Pseudomonas and Serratia previously identified using 16S rRNA [28] were studied for assessing their metal and antibiotic tolerance properties. Minimum inhibitory concentration of the four heavy metals Pb, Zn, Cu, and Cd towards bacterial isolates was determined by spot inoculation technique as described previously [29]. Stock solutions (10,000 ppm) of metals were prepared from their respective metal salts of zinc sulfate [ZnSO4·H2O], lead nitrate [Pb(NO3)2], copper sulphate [CuSO4·5H2O] and cadmium nitrate [Cd(NO3)2]. The working solutions of heavy metal concentrations were prepared from the stock to achieve the final concentration ranging from 50 to 2000 ppm for Pb, Zn, Cu, and 1–100 ppm for Cd in the media. The isolates were grown overnight in Luria–Bertani broth and 10 µl of the culture corresponding to OD 0.5 at 600 nm was spotted on the metal containing plates and incubated at 28 °C for 24 h. Minimum inhibitory concentration was determined as the lowest concentration of the metal that inhibited growth after 24 h [30].

Antibiotic Susceptibility Test for the Isolates

The antibiotic susceptibility of the bacterial isolates was conducted using agar diffusion technique on Mueller–Hinton Agar (MHA) plates following the guidelines of the Clinical Laboratory Standard Institute (CLSI), 2013. In the present study, a total of 15 antibiotics were used viz, Meropenem (10 mcg), Imipenem (10 mcg), Erythromycin (10 mcg), Ciprofloxacin (5 mcg), Nalidixic acid (30 mcg), Netillin (30 mcg), Novobiocin (30 mcg), Neomycin (30 mcg), Tigecycline (15 mcg), Lincomycin (15 mcg), Rifampicin (5 mcg), Polymyxin B (300U), Co-Trimoxazole (25 mcg), Ceftazidime (30 mcg), Trimethoprim (5 mcg) using standard antibiotic discs (HiMedia, India). The plates were observed for a clear inhibition zone after incubation for 24 h at 28 °C and the diameter of the zone was measured with a HiAntibiotic Zone Scale™-C (HiMedia, India) [31]. The MAR index for the test isolates was calculated as a/b where a represents the number of antibiotics the isolates are resistant to and b represents the total number of antibiotics used against the isolate as described previously [32]. A MAR index value of  >  0.2 indicates that the isolates are multiple antibiotic-resistant whereas a MAR index value of < or = 0.2 indicates a very low or negligible antibiotic resistance [32].

Screening of Heavy Metals and Antibiotic Resistance Genes in the Bacterial Isolates

Metal-transporting genes belonging to PIB-type ATPase and RND families were screened using specific forward and reverse primers as indicated in Supplementary 1. Similarly, the efflux system belonging to the RND family of proteins mainly involved in antibiotic resistance in bacteria was screened using the respective forward and reverse primers listed in Supplementary Table 1. The primers were designed from National Centre for Biotechnology Information (NCBI) using the primer designing tool. The screening of the genes was done from the bacterial genomic DNA extracted using the Bacterial Genomic DNA extraction kit (HiMedia, India). A few isolates from the genera Pseudomonas and Serratia were chosen for the screening and profiling of heavy metal and antibiotic resistance genes due to their elevated MAR index and prevalence across the sampled areas. Polymerase chain reaction (PCR) was carried out with 25 µl reaction volume containing 2 µM each of forward primer and reverse primer, 10X buffer with 17.5 mM MgCl2, 250 µM each of dATP, dCTP, dGTP, and dTTP, 1.0 U of Taq DNA polymerase and 30 ng of the DNA template. The amplification steps involved denaturation at 95 °C for 5 min, followed by 30 cycles with denaturation at 94 °C for 1 min, annealing was done at specific temperatures for each primer set provided in Supplementary Table 1 for 1 min, and extension at 72 °C for 1.5 min followed by a final extension step of 72 °C for 5 min using a GeneAmpH PCR system 9700 (Applied Biosystems, USA). The amplicons were purified using Gel Extraction Kit (HiMedia, India) and then sequenced.

Sequencing and Phylogenetic Analysis of the Genes

The amplified genes were electrophoresed on agarose gel (1.5%) followed by purification using the HiMedia Gel Extraction Kit (HiMedia, India) and sequenced from AgriGenome Labs, Kerala, India using Abi 3730XL Genetic Analyzer, USA. The nucleotide sequences obtained were subjected to homology analysis using sub tool (BLASTX) in Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov) to determine the phylogenetic neighbours of the respective genes against the GenBank database, NCBI, USA. A molecular phylogenetic tree was constructed using the Neighbour-joining method in MEGA v 7.0 [33] with 1000 bootstrap replicates for nodal support.

Sequence Accession Numbers

Nucleotide sequences of 16S rRNA genes of the previously published isolates were obtained from EzBioCloud database (http://www.ezbiocloud.net). The relevant sequences of the bacterial isolates described in the present study have been deposited in the NCBI database and Accession Numbers obtained as MN733215, MN733339, MN727121, MN727123, MN733345, OK090518, OK090542, OK090597, MN733233, MN733224, MN733225, MN733340, MN733087.

Results

Heavy Metal Tolerance and Antibiotic Sensitivity of the Isolates

In this study, MIC values and MAR index were recorded and reported from our laboratory for hundred and seven (107) bacterial isolates post tolerance tests to 4 heavy metals (Pb, Zn, Cu, Cd) and 15 antibiotics [28]. Among these isolates, the Pseudomonas sp. and Serratia sp. were found to tolerate a wide range of heavy metal concentrations and antibiotics evinced as by high MIC and MAR values, respectively (Table 1). Hundred percent of both species displayed uniform MIC values of 2000 and 500 ppm for Pb and Cu, respectively. While only 58% of Pseudomonas versus 100% Serratia species displayed 100 ppm MIC for Cd. Conversely, the MIC for Zn evinced large variation, with values of 500 and 1000 ppm for 39% of all Pseudomonas and 80% of Serratia species, respectively.

Table 1 MIC and MAR index values of the studied isolates
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Occurrence of Metal Transporting PIB-Type ATPase and RND Genes

Only Pseudomonas and Serratia isolates with high MIC and MAR values against metals and antibiotics, respectively, were further investigated to detect RND and PIB-type ATPase genes. zntA/cadA/pbrA genes belonging to PIB-type ATPase and czcA genes of RND were successfully amplified in Pseudomonas and Serratia species (Fig. 1). The occurrence of metal transporting genes from RND and PIB-type ATPase families were screened using primers that were either designed as required or utilized from the previously described sources [34,35,36]. Nine Pseudomonas isolates showed amplifiable PCR products for the PIB-type ATPase gene and four isolates for RND efflux proteins. Among them, WRK8 and ST3 were found to be PCR positive for both PIB-type and RND proteins; whereas five Serratia isolates were found to carry PIB-type ATPase gene (Table 2). The phylogenetic clustering with the PCR amplified gene sequences corresponded to metal translocating PIB-type ATPases, and cusA/czcA belonging to RND protein family, as depicted in Fig. 2a and b, respectively.

Fig. 1
figure 1

Electrophoresis gel picture of the amplified genes. a shows the amplification of czcA gene; lane 1–100 bp ladder, lane 3, 5, 9, 10 and 11 shows the amplification of czcA gene in Pseudomonas species. b shows the amplification of mexY and mexF genes; lane 1–100 bp ladder, lane 2 and 3 shows the amplification of mexY and mexF genes in Pseudomonas species. c shows the amplification of PIB-type ATPase gene; lane 1–100 bp ladder; lane 2, 3 and 4 shows the amplification of zntA/cadA/pbrA gene in Pseudomonas species and lane 5, 6 and 7 shows amplification of zntA gene in Serratia species. d shows the amplification of sdeB genes; lane 1–100 bp ladder, lane 2 and 3 shows the amplification of sdeB genes in Serratia species

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Table 2 BLASTX analysis of metal transporting and multidrug efflux pump genes
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Fig. 2
figure 2

Phylogenetic analysis of the PIB-type ATPase zntA/cadA/pbrA (a) and RND czcA (b) metal transporting genes in the representative isolates. Neighbor-joining method of translated amino acid sequences was carried out using MEGA v7.0 software with 1000 bootstrap replications

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Similarly, considering the resistance of the isolates towards various classes of antibiotics, the isolates were further screened for the presence of efflux pumps belonging to the RND family of proteins. Pseudomonas species were screened for mexB, mexF and mexY genes while Serratia species were screened for the presence of sdeB genes. Subsequently, Pseudomonas WRn10 harboured both mexF and mexY genes; while the isolate ST3 exhibited presence of both mexB and mexF genes, whereas the WC4 isolate contained only mexF gene. While the Serratia isolates WT6, WT7 and WT17 contained only the sdeB genes. The BLAST analysis of the gene amplicons showed 100% similarity with the RND transporter permease from various operons like MexAB-OprM, MexEF-OprN, MexXY-OprM, SdeAB operons (Table 2); while the genes corresponded to the multidrug efflux RND transporter permease (Fig. 3).

Fig. 3
figure 3

Phylogenetic analysis of the multidrug efflux pump genes namely, sdeB, mexB, mexF and mexY in the representative isolates. Neighbor-joining method of translated amino acid sequences were carried out using MEGA v7.0 software with 1000 bootstrap replications

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Evidence of Horizontal Gene Transfer of PIB-type ATPase Among the Isolates

Phylogenetic incongruency between the studied gene and a marker gene (the conserved 16S rRNA gene) along with GC content analysis has been used to study the HGT of PIB-type ATPases amongst bacteria [27, 34]. The sequences of zntA/cadA/pbrA gene obtained from the isolates were analyzed by BLASTX to locate the closest relative from the NCBI database. The zntA/cadA/pbrA gene sequences from Pseudomonas WC7 and WC9 showed less than 50% similarity to PIB-type ATPase from Pseudomonas sp., whereas they showed 100% similarity to the PIB-type ATPase genes from Bacillus species. The clustered configuration in the phylogenetic analysis with 100% bootstrapping indicated possible inter-phylum evidence of HGT (Fig. 4). Moreover, GC content analysis of PIB-type gene from these isolates was around 38% which deviates from the normal GC content of Pseudomonas sp. (60–68%), but is very similar to GC content of Bacillus sp. (32–38%) (Table 3). These findings indicated that a possible intergeneric HGT that has developed in these isolates.

Fig. 4
figure 4

Neighbor-joining analysis of heavy metal transporters zntA/cadA/pbrA PIB-type ATPase gene (a) and 16S rRNA gene (b) of the isolates. The accession number of the respective genes are provided in the brackets. PIB-type positive isolates showing possible HGT are highlighted and connected by lines

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Table 3 Comparative matches for the closest phylogenetic neighbors obtained for the isolates based on 16S rRNA gene and PIB-type ATPases gene profiling
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Discussion

The presence of heavy metals in the environment and the release of bioactive compounds from different microorganism causes selective pressure leading to an increase in bacterial resistance to heavy metals and antibiotics [28, 37]. Metals like Pb, Zn, Cu, and Cd that have multifaceted and extensive applications in industries enter the environment variously via vehicular emissions, batteries, mining activities and gasoline spillage [38]. Similarly, the rampant and continuous use of antibiotics have given rise to the evolution of multi drug resistant strains [11].

Bacterial resistance to different metals could be explained either by the mechanisms of co-resistance or cross resistance; where genetically linked factors in close physical proximity are expressed simultaneously by co-resistance; whereas the same factor or gene via control resistance lead to generation of several antimicrobials by cross resistance [13, 39, 40]. The role of cross resistance has been documented in earlier studies in which plasmid carrying metal and antibiotic resistance genes from one bacteria have been successfully expressed in recipient strains [41]. Similarly, cross resistance has been shown to play a role in antibiotic resistance and metal tolerance through common efflux systems [40].

Efflux proteins from RND and PIB-type ATPase families are known to confer metal tolerance in bacteria [7]. In the present study, the occurrence of efflux proteins from RND and PIB-type ATPase families in the isolates endorsed survival in metal-contaminated areas. The transmembrane efflux proteins can translocate heavy metals like Pb, Zn, Cu, and Cd from the intra cytoplasmic region to the periplasm or even cell exterior [9]. Additionally, some Pseudomonas species were found to harbor both cusA/czcA gene which encode efflux proteins belonging to the RND family and PIB-type ATPase family of proteins. Similar studies have documented the occurrence of metal transporter genes and antibiotic resistance genes in Pseudomonas species [42]. The czc system is a cobalt/zinc/cadmium resistance determinant that was first studied in the Ralstonia sp. In this system, CH34 is a trans-envelope transporter that uses proton gradients to extrude metal cations from the cell, as opposed to the ATP driven PIB-type ATPase [43].

Multidrug resistance is a global health concern that could escalate due to rampant antibiotic use and exposure of microorganisms to various antimicrobials agents [44, 45]. The presence of various industries especially pharmaceutical companies near the riverine ecosystems could raise the frequency of resistant strains to various antimicrobials [46]. Multidrug efflux pumps are known to contribute to the intrinsic resistance of these bacteria to several classes of antibiotics like quinolones, chloramphenicol, beta-lactams, tetracyclines, novobiocin, and macrolides [21]. In this study, chemiosmotic multidrug efflux pumps that provide resistance to several antimicrobials (antibiotics, heavy metals, and biocides) encoded by sdeAB in Serratia and mexAB, mexEF, or mexXY in Pseudomonas were detected [47]. In addition, isolates having greater tolerance to Cd displayed a very high MAR index value (0.53 for Pseudomonas and 0.46 for Serratia) and Cd emerged the most toxic metal in the present investigation. Previous reports not only corroborate efflux pumps engendering the high MAR index in some isolates, but endorse Cd as the most toxic metal [48, 49].

Mobile genetic elements such as plasmids and integrons that encode several resistance determinants can translocate among microorganisms via HGT forging greater antimicrobial resistance and metal tolerance [1]. Horizontal gene transfer is a well-known route for the evolution of new traits amongst microorganisms especially in metal-contaminated sites [34]. Analysis of the translated amino acid sequences of PIB-type ATPase genes (zntA/cadA/pbrA loci) in this study by the neighbor-joining and maximum likelihood method revealed noteworthy incongruence between the ATPase gene and 16S rRNA phylogeny as reported in earlier studies [27, 34]. In this study, two PIB-type ATPase amplicons from Pseudomonas sp. of phylum Proteobacteria showed 100% similarity to Bacillus sp. from the NCBI database, and were clustered with the PIB-type ATPase of Bacillus from phylum Firmicutes. The indication of HGT occurrence at an inter-phylum level after the initial analysis was bolstered by comparison of G + C content. It revealed the inter-phylum presence of the PIB-type ATPase genes between Bacteroidetes and Firmicutes [27]. Hence, the current study highlights the occurrence of metal and antibiotic-resistant isolates harboring metal and antibiotic resistance genes from the Teesta riverine ecosystem which can serve a reservoir for exchange or movement of these gene in the environment. However, the study of the occurrence of these genes in other bacterial species and/or metagenome from these sites would lend clarity and in-depth understanding to the prevalence and dissemination of such genes in the habitat.

Conclusion

Predominant Pseudomonas and Serratia isolates from the Teesta River, a once pristine river, showed a wide range of metal and antibiotic resistance property that might have evolved from exposure to high metals and antibiotics present in their habitat. These isolates are equipped with metal and antibiotic resistance genes that are either inherent in their genome and/or have been acquired through lateral movement of genes, as evident from the present study. Furthermore, riverine ecosytems that are exposed to pharmaceutical and metal-related industries can act as incubators for the evolution of multi drug resistance strain bacteria. The findings indicated that the bacteria can adapt to their extant environment by altering their altered phenotype, which can be an important tool to study the emergence of metal tolerant strains, especially in antibiotic resistance traits that are clinically important.