Overproduction of Efflux Pumps as a Mechanism of Metal and Antibiotic Cross-Resistance in the Natural Environment
1
Faculty of Natural Science, University of Ss. Cyril and Methodius in Trnava, Nam. J. Herdu 2, 917 01 Trnava, Slovakia
2
Faculty of Natural Science, Pavol Jozef Safarik University in Kosice, Srobarova 2, 041 54 Kosice, Slovakia
3
Institute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, 040 01 Kosice, Slovakia
*
Author to whom correspondence should be addressed.
Sustainability 2023, 15(11), 8767; https://doi.org/10.3390/su15118767
Submission received: 5 April 2023
/
Revised: 25 May 2023
/
Accepted: 27 May 2023
/
Published: 29 May 2023
(This article belongs to the Special Issue Interaction of Microorganisms with Metals and Minerals)
Abstract
:Antibiotic and metal resistance can occur together in the environment and can be linked by the same detoxication mechanism (cross-resistance). The understanding of this linkage may be a key to further study of the spread of antibiotic resistance in the non-hospital environment worldwide. In our study, we examined the overproduction of efflux pumps as a possible mechanism of the cross-resistance of isolates originating from industrial and mine tailings. Resistance to metals (Cu, Ni, Zn and Pb) and antibiotics (ampicillin, chloramphenicol, tetracycline and kanamycin) was observed at all the sampling sites and ranged from 16 to 75%. Multiresistance (MAR index > 0.38) was recorded in 26% of the isolates and was associated with the metal selective pressure duration. Overproduction of efflux pumps has mainly been observed in multiresistant isolates. Our results may indicate that the overproduction of efflux pumps could be the mechanism of cross-resistance between metals and therefore related to metal and antibiotic multiresistance. The results also show that the importance of sustainably storing metal-containing waste lies not only in its environmental impact but also in human health via antibiotic resistance proliferation.
1. Introduction
The importance of sustainability in metal-containing waste storage and waste management lies in mitigating the harmful effects of improper disposal and preserving the environment for future generations [1]. The use of metals in various industries, including mining, agriculture and manufacturing, has led to the release of metals into the environment, causing harm to ecosystems and human health. The adverse effects of metals are not limited to their direct impact on the environment. In addition to their direct toxic effects on plants and animals, metals can also have indirect effects on the microbial communities that play important roles in maintaining ecosystem health. Sustainable storage practices for metal-containing waste, such as proper waste management and recycling of metals, are crucial for reducing metal contamination and minimizing the risks associated with metal exposure [2]. In addition to metal storage, metals can be removed from the environment using bacterial bioleaching. One of the key benefits of using bacteria for metal removal is that it is often a more sustainable approach than traditional methods such as chemical treatment or excavation. Bioremediation is a natural process that does not require the use of harsh chemicals or heavy machinery, reducing the environmental impact of the remediation process.
Furthermore, bacteria can often be used to remove metals in situ, meaning that the contaminated material does not need to be excavated and transported to another location for treatment. Therefore, the study of bacteria involved in metal strains has wide-ranging applications, both in pollution monitoring and its possible removal. [2,3].
Metal contamination has emerged as a pressing environmental issue, and its indirect consequences are still not well-understood [2]. The accumulation of metals in the environment creates a constant selective pressure that can impact microbial communities in various ways [3]. Several studies have indicated that we are on the verge of a post-antibiotic era, making the treatment of multiple infections challenging or even impossible [4,5]. Investigating the role of metals in the proliferation of antibiotic-resistant genes in the environment could be a key factor in slowing down this process. However, it is still unclear how significant the metal contribution is in the spread of antibiotic resistance under natural conditions compared to clinical environments.
Some mechanisms by which bacteria directly defend themselves against the effects of metals and antibiotics are more common than others. Chen et al. [6] pointed out that the main mechanisms of resistance to macrolide–lincosamide–streptogramin B (MLSB) and aminoglycosides are antibiotic deactivation (42%), overproduction of efflux pumps (38.7%), cellular protection mechanisms (11.8%) and other mechanisms (7.5%). Efflux pumps are considered one of the common mechanisms of cross-resistance to metals and antibiotics [7].
Recent studies have also shown a connection between the presence of toxic metals in soil or water environments and the transmission of antibiotic-resistance genes [3,8,9]. While most of these studies have focused on agricultural soil [9,10], soil near mines [3] or hospital and natural wastewater [10,11], only a few have investigated the ore itself [12], and those have been limited to specific locations without comparisons. Therefore, it is essential to compare different ore sampling sites to confirm the presence of antibiotic and metal resistance at these contaminated sites, which are not limited to one specific type of metal or ore.
The term cross-resistance refers to a mechanism that simultaneously reduces bacterial susceptibility to substances with similar structures or functions [13] and is often the result of a mutation [14]. Cross-resistance can be found between two different antibiotics, between antibiotics and biocides and between antibiotics and metals [15]. Mechanisms responsible for cross-resistance can vary from active efflux or disulphide bond formation to internal or external sequestration and transformation of the compound into a less toxic form [16]. Nowadays, there are several studies documenting cross-resistance to metals and antibiotics in clinical environments and agricultural areas [3,17,18].
Some resistance mechanisms are more likely to be responsible for metal and anti-biotic cross-resistance than others [16]. According to Berendonk et al. [17], environmental pollution by metals usually leads precisely to an increased incidence of gene-encoding efflux pumps. Efflux pumps are known to be able to exclude several antibacterial agents from bacteria in a very short time, such as antibiotics and metals [19,20]. The cross-resistance is based on the co-regulation process, which is activated when higher concentrations of metals or antibiotics are detected, and the transduction cascade of the same defence mechanism is triggered. This mechanism can lead to the co-expression of antibiotic and metal-resistant genes at the same time [21].
The overproduction of efflux pumps as a bacterial defence mechanism against metals and antibiotics has been studied mostly in a clinical environment [16,21]. However, the spread of antibiotic resistance is not limited to the clinical environment, and the natural environment is a vast, often unexplored area where it can spread and be maintained through other selection factors. This phenomenon has been little studied in natural environments, where only the selection pressure of metals is present without the direct selection pressure of antibiotics. Among those environments, the mining environment and industrial landfills represent a potentially dangerous environment for the spread of antibiotic resistance through the overproduction of efflux pumps due to the high content of metals as a selection factor [20].
Additionally, the co-location of efflux pump production genes on mobile genetic elements facilitates their dissemination alongside other co-selected traits in the environment, increasing the potential for resistant gene transfer. This interconnected transfer of genetic elements not only reinforces the co-selection phenomenon but also poses a significant challenge to the control and mitigation of antibiotic resistance. The utilization of metal-resistant genes in co-selection not only contributes to the spread of antibiotic resistance but also sustains its persistence across diverse human-utilized environments, amplifying the complexity of addressing this urgent global health concern [21].
Our work aimed to demonstrate the overproduction of efflux pumps as a mechanism of cross-resistance of metal- and antibiotic-resistant isolates from metal-contaminated environments. The study of the possible effects of metal dumps on antibiotic resistance proliferation can bring new light to the importance of sustainability in metal storage and its removal. Implementing sustainable waste management practices is a critical step towards reducing not only the environmental impact of metal storage but also its impact on the bacterial community and even on human health.
This paper’s innovation lies in enhancing our comprehension of the interrelationship between surface and deep underground metal-contaminated sites. Additionally, it investigates the potential mechanism of cross-tolerance between metals and antibiotics, along with other contributing factors. It is imperative to acknowledge metal dumps not solely in terms of their environmental toxicity, but also as a significant source of selection pressure for the proliferation of antibiotic resistance. This issue is presently a prevailing concern adversely impacting human health.
The ethidium bromide-agar method used in our experiment to determine the overexpression of the bacterial efflux system is also used for detecting phenotypes responsible for multidrug resistance recognition. This method is based on the existence of a maximum concentration of ethidium bromide, which is effectively extruded by the cell’s efflux system, with the assumption that higher concentrations of ethidium bromide will be captured in the cells of susceptible bacteria. Subsequently, when this bacterial mass is exposed to UV light, fluorescence will be detected [22,23].
2. Materials and Methods
2.1. Description of the Sampling Sites and Sample Withdrawal
Two industrial and mine landfills located in Slovakia (Hodruša-Hámre and Košice) were investigated. Ore samples were withdrawn from three sampling sites at Hodruša: one surface landfill (H/L) and two deep underground (500 m) sampling sites (H/U1, H/U2), in addition to from one surface landfill at Košice (K/L). Soil samples were withdrawn only from the Hodruša sampling site (H/soil). Under sterile conditions and at 4 °C, the soil samples were obtained and transported to the laboratory for immediate bacterial isolation. The concentrations of both metallic and non-metallic elements were analysed by drying and crushing 10 g of each sample to a minimum grain size of 160 microns, followed by measurement using an XRF analyser (X-Ray Fluorescence Analyser, Delta Premium 50, Olympus, Tokyo, Japan) in the “light matrix” mode. The pH levels of all the sampling points were determined according to standard STN ISO 10390. The ore processing and soil withdrawal were described in more detail in previous studies [24,25].
The main difference between the Košice and Hodruša localities is the chemical composition of the ore and the different levels of the processing process. The Košice landfill is constantly refilled with ore waste, in comparison to Hodruša, where ore arrives from the mine and at the landfill before industrial processing. At the Hodruša sampling site, the landfill is composed of sulfidic ore that was extracted directly from the mine before processing.
The location and main characteristics of the sampling sites are shown in Figure 1.
2.2. Isolation, Selection and Characterisation of the Bacterial Population
One-half gram of the ore and soil samples was resuspended in phosphate-buffered saline (PBS), and aliquots were plated on a TSA medium (Tryptone Soya Agar, Oxoid, Thermo Fisher Scientific, Waltham, MA, USA) and cultivated under aerobic conditions at room temperature for 48–72 h. The standard protocol for isolation and identification was followed in this study, which was recommended by Bruker Daltonics. The protocol can be found in the Bruker Guide to MALDI Sample Preparation–MALDI Preparation Protocols (2012), available online (https://researchservices.pitt.edu/sites/default/files/Bruker_Guide%20for%20MALDI_Sample_Preparation.pdf (accessed on 29 March 2023)). Isolates from Hodruša were subjected to MALDI–TOF analysis (matrix-assisted laser desorption ionisation–time of flight) as part of a previously published thesis [18]. To select isolates for further analysis, a dendrogram was obtained, and the isolates were chosen to represent individual branches in the dendrogram. The selected isolates accounted for 22–30% of the total number of bacteria isolated from each sampling site.
2.3. Metal and Antibiotic Resistance Testing
Minimum inhibitory concentrations of metals (Cu, Pb, Fe, Ni and Zn,) and antibiotics (chloramphenicol, ampicillin, kanamycin and tetracycline,) were evaluated by the dilution method on solid Müller–Hinton media with the selected antibiotic/metal concentrations (Table 1), according to the modified test of Aleem et al. [26]. The first concentration of metal/antibiotic which prevents bacterial colonies from growing was considered the minimum inhibitory concentration. This passage outlines the approach used to evaluate the resistance of bacteria to antibiotics and metals in two different locations. The concentration of antibiotics was chosen following EUCAST standards, but the testing range was widened for certain locations where the bacteria were either more sensitive or more tolerant. The selection of antibiotics and metals for testing was based on their widespread usage and propensity for resistance. The dilution method was used to assess antibiotic resistance on Müller–Hinton agar, and a modified test was used to evaluate metal resistance. The minimum inhibitory concentration (MIC) was established as the concentration at which the bacteria ceased forming visible colonies. Bacterial isolates that could survive at a particular concentration of antibiotics or metals were deemed to be tolerant to that level.
From the minimum inhibitory concentration, we evaluated resistance to tested antibiotics and metals. Isolates were considered resistant to metals/antibiotics if capable of forming a visible colony after exposure to concentrations of the metals/antibiotics listed in Table 2. The limit for antibiotic resistance evaluation was obtained by increasing the resistance limit for pathogenic bacteria set by EUCAST (http://www.eucast.org (accessed on 29 March 2023)). Bacteria were considered resistant to metals if they were able to form visible colonies after exposure to the second-lowest concentrations of each of the tested metals.
2.4. Determination of Multi-Antibiotic Resistance
We assessed the occurrence of multiresistance by using the MAR index, which we obtained by following the calculation (Equation (1)) [3]. A bacterium that is resistant to more than half of the antibiotics tested is considered multiresistant, and thus its MAR ≥ 0.5.
where r is the number of bacteria to which the bacterium is resistant, and A is the total number of tested antibiotics.
MAR = r/A
2.5. Determination of Overproduction of Efflux Pumps
To determine the overproduction of efflux pumps, the methodology according to Martis et al. [23] was followed. Plates were prepared by serial dilution with TSB medium containing a decreasing concentration of ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA). Concentrations of ethidium bromide at 0.5, 1, 1.5, 2 and 2.5 mg/mL were tested. The cultures of bacteria were incubated for 2 to 5 days at room temperature in the dark. The overproduction of efflux pumps was evaluated by observing fluorescence after UV exposure. The higher the concentration that had to be used to induce fluorescence, the greater the overproduction of efflux pumps. The isolate was considered as an isolate with efflux pump overexpression when fluorescence was observed for the first time at a concentration of 1 μg/mL ethidium bromide in the medium, according to Aleem et al. [26].
3. Results and Discussion
3.1. Antibiotics and Metal Resistance
Resistance to metals and antibiotics has been reported at all the sampling sites and ranges from 16 to 74% of the isolates, depending on the tested antibiotic/metal (Table 3). The MIC values of the most resistant bacteria were 800 μg/mL for ampicillin, 50 μg/mL for chloramphenicol, 50 μg/mL for tetracycline, 200 μg/mL for kanamycin, 1000 μg/mL for zinc, 500 μg/mL for copper, 750 μg/mL for nickel and 2000 μg/mL for lead.
While isolates taken from Hodruša showed resistance to almost all metals, isolates from Košice mainly showed resistance only to lead. According to our previous results, multiresistance was more common at the Hodruša sampling site [24,25], where isolates were resistant to 1–3 antibiotics compared with isolates from Košice, which were mainly resistant to kanamycin and, rarely, to tetracycline.
The different levels of resistance between the sampling sites could be mainly related to the chemical composition of the sampling sites and the metal exposure time. Both sampling sites for industrial metal processing contained a high concentration of metals, but the metal composition was different. The Košice landfill is refilled continuously with ore waste after metal processing involving high temperatures, so we could say that the effect of metal on the present microorganisms was short-term. Moreover, the Hodruša landfill contains ore before processing, and the microbial community should be the same as in the deep mine sampling site with a long-term selection pressure of metals. We assumed that the long-term selection pressure of metals on the present microorganisms at Hodruša may cause a higher multiresistance presence.
Moreover, the metals at the Hodruša sampling site were introduced through mining and primary processing of ore, while, at the Košice site, the landfill was composed of waste from blast furnace metal processing. The chemical composition of the waste material played a role in the findings, with the Košice site containing oxide slag that had lower solubility, which could reduce the number of metals released into the environment and make them less toxic. The nature of the sites could also impact antibiotic resistance, as Košice is an active industrial landfill that is continually filled, whereas Hodruša is partially open.
3.2. Overproduction of Efflux Pumps
The absence of fluorescence, which should be the evidence of overproduction of efflux pumps, was mainly observed in multiresistant isolates from Hodruša with a MAR index greater than 0.38 (Table 4). In contrast, isolates sensitive to multiple antibiotics/metals were unable to eliminate even low levels of ethidium bromide from their cells. An exception is the NRI-11 isolate, which exhibits an overproduction of efflux pumps but is not multiresistant to the tested antibiotics and metals. However, our experiment did not disprove the possibility that this isolate may be multiresistant to other antibiotics or metals not included in our testing.
In the case of two multiresistant isolates (Acidovorax spp. and Rhodococcus erythropolis), we did not find a concentration of ethidium bromide that caused fluorescence, which means that the isolates could exclude every tested concentration of ethidium bromide out of the cell. This may indicate a significant overproduction of efflux pumps compared to other isolates. According to our results, the overproduction of efflux pumps can be the mechanism of cross-resistance and can be related to the level of multiresistance of individual bacterial isolates.
However, the overproduction of efflux pumps is probably not the main mechanism of antimicrobial resistance to lead and kanamycin, which was frequent at the Košice sampling site. Nevertheless, the efflux pumps are a natural part of both prokaryotic and eukaryotic cells [28], so their contribution to the elimination of lower concentrations of toxic substances cannot be excluded. One important factor that could affect the detection of efflux pump overproduction and could also be tested is the type of inducer. Ethidium bromide may not always be a suitable inducer; therefore, to further investigate efflux pump presence, it would be appropriate to examine the effects of sublethal concentrations of other inducers. Examples of the evaluation of fluorescence are shown in Figure 2.
Authors in a similar study [29], who used the same methodology but tested it on clinical isolates of representatives of the genus Pseudomonas, showed fluorescence regardless of their resistance level to metals or antibiotics. We explain these results by the natural ability of representatives of this genus to autofluorescence when exposed to direct UV light [30,31]. Resistance mediated by efflux pumps is more common in some bacterial genera than in others. In the bacterial genus Pseudomonas, efflux pumps occur as part of their natural resistance to many antibiotics, including some β-lactam antibiotics (aminopenicillins, first- and second-generation cephalosporins and aminopenicillins) [32,33].
As in our work, Shinde and Thombre [34] examined several Gram-positive and Gram-negative bacteria isolated from the natural environment. The minimum inhibitory concentration (MIC) for most resistant isolates in this study was 1 mg/L of ethidium bromide, which the authors considered evidence of the overproduction of efflux pumps. In contrast to these results, some of the bacteria studied by us were able to eliminate even twice that concentration. This difference can be caused by the different environments from which the bacteria were isolated or by the different multiresistance profiles. Thus, our results could indicate that the presence of metals in the environment leads to increased excretion of antimicrobials (antibiotics and metals) out of bacteria through efflux pumps, as Rajbanshi [7] and Seiler and Berendonk [19] assumed in their studies. According to our results, the overproduction of efflux pumps could be the mechanism of metal and antibiotic cross-resistance and could be related to the level of multiresistance of individual bacterial isolates.
4. Conclusions
The issue of antibiotic and metal multiresistance in metal-contaminated areas is a major concern for public health and environmentalists. This study contributes to the existing body of knowledge by providing evidence for the occurrence of antibiotic and metal multiresistance in such areas. The presence of multiresistance was found to be positively associated with the duration of metal exposure, which highlights the need for sustainable storage practices for metal-containing materials to prevent long-term environmental and human health consequences. This finding is particularly important, as metal contamination and its impact on human health and the environment have been a growing concern worldwide.
The study found that there was a significant level of resistance to metals and antibiotics across all the sampling sites, ranging from 16 to 74% of isolates, with the most resistant bacteria having MIC values of 800 μg/mL for ampicillin, 50 μg/mL for chloramphenicol and tetracycline, 200 μg/mL for kanamycin and 1000 μg/mL for zinc. The Hodruša sampling site showed more multiresistance, with isolates resistant to one to three antibiotics compared to isolates from the Košice site, which were mainly resistant to kanamycin and, rarely, to tetracycline. Differences in metal composition and exposure time could be the reason for the different levels of resistance between the sampling sites. The long-term selection pressure of metals at Hodruša may cause a higher multiresistance presence, while the Košice landfill, which is continuously filled with ore waste after metal processing, may have a short-term effect on metal-exposed microorganisms.
It is clear from the study that the overproduction of efflux pumps could be a common mechanism of cross-resistance, which is related to the level of multiresistance. This finding is crucial, as efflux pumps play a crucial role in the survival of bacteria in metal-contaminated environments. Further research is needed to gain a deeper understanding of the processes of cross-resistance to metals and antibiotics mediated by efflux pumps in polluted environments. This knowledge is essential for the development of effective strategies to prevent and manage the spread of antibiotic and metal multiresistance.
In conclusion, this study has provided valuable insights into the occurrence of antibiotic and metal multiresistance in metal-contaminated areas. The results emphasise the importance of adopting sustainable methods for storing materials containing metals to avoid any long-term harmful impacts on the environment and human health. Further research is necessary to gain a deeper understanding of the mechanisms of cross-resistance to metals and antibiotics mediated by efflux pumps in polluted environments. The implementation of effective strategies to prevent and manage the spread of antibiotic and metal multiresistance is essential for safeguarding public health and the environment.
Author Contributions
M.S. wrote the paper. J.S.-K. and A.L. conceived and designed the analysis. M.S. and K.Š. performed the MIC analysis and efflux pump experiments. M.S., J.S.-K. and A.L. edited the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding
The work was supported by financial aid from Slovak Grant Agency (project No. VEGA 1/0018/22).
Data Availability Statement
Not applicable.
Conflicts of Interest
The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript or in the decision to publish the results.
References
- Huang, Y.; Wang, L.; Wang, W.; Li, T.; He, Z.; Yang, X. Current status of agricultural soil pollution by heavy metals in China: A meta-analysis. Sci. Total Environ. 2018, 651, 3034–3042. [Google Scholar] [CrossRef] [PubMed]
- Mandal, M.; Das, S.N.; Mandal, S. Principal component analysis exploring the association between antibiotic resistance and heavy metal tolerance of plasmid-bearing sewage wastewater bacteria of clinical relevance. Access Microbiol. 2020, 2, e000095. [Google Scholar] [CrossRef] [PubMed]
- Sinegani, A.A.S.; Younessi, N. Antibiotic resistance of bacteria isolated from heavy metal-polluted soils with different land uses. J. Glob. Antimicrob. Resist. 2017, 10, 247–255. [Google Scholar] [CrossRef]
- Silvester, R.; Madhavan, A.; Kokkat, A.; Parolla, A.; Adarsh, B.M.; Harikrishnan, M.; Abdulla, M.H. Global surveillance of antimicrobial resistance and hypervirulence in Klebsiella pneumoniae from LMICs: An in-silico approach. Sci. Total Environ. 2021, 802, 149859. [Google Scholar] [CrossRef]
- Kwon, J.H.; Powderly, W.G.; Power, S.; Lengaigne, M.; Saitou, M.; Hayashi, K.; Swaney, D.L.; Ramms, D.J. The post-antibiotic era is here. Science 2021, 373, 471. [Google Scholar] [CrossRef] [PubMed]
- Chen, J.; Li, J.; Zhang, H.; Shi, W.; Liu, Y. Bacterial heavy-metal and antibiotic resistance genes in a copper Tailing Dam Area in Northern China. Front. Microbiol. 2019, 10, 1916. [Google Scholar] [CrossRef] [PubMed]
- Rajbanshi, A. Study on Heavy metal resistant bacteria in Guheswori sewage treatment plant. Our Nat. 2009, 6, 52–57. [Google Scholar] [CrossRef]
- Zagui, G.S.; Moreira, N.C.; Santos, D.V.; Darini, A.L.C.; Domingo, J.L.; Segura-Muñoz, S.I.; Andrade, L.N. High occurrence of heavy metal resistance genes in bacteria isolated from wastewater: A new concern? Environ. Res. 2020, 196, 110352. [Google Scholar] [CrossRef]
- Salam, L.B. Unravelling the antibiotic and heavy metal resistome of a chronically polluted soil. 3 Biotech 2020, 10, 238. [Google Scholar] [CrossRef]
- Peng, S.; Dolfing, J.; Feng, Y.; Wang, Y.; Lin, X. Enrichment of the antibiotic resistance gene tet (L) in an alkaline soil fertilized with plant derived organic manure. Front. Microbiol. 2018, 9, 1140. [Google Scholar] [CrossRef]
- Dickinson, A.; Power, A.; Hansen, M.; Brandt, K.; Piliposian, G.; Appleby, P.; O’Neill, P.; Jones, R.; Sierocinski, P.; Koskella, B.; et al. Heavy metal pollution and co-selection for antibiotic resistance: A microbial palaeontology approach. Environ. Int. 2019, 132, 105117. [Google Scholar] [CrossRef] [PubMed]
- Ji, X.; Shen, Q.; Liu, F.; Ma, J.; Xu, G.; Wang, Y.; Wu, M. Antibiotic resistance gene abundances associated with antibiotics and heavy metals in animal manures and agricultural soils adjacent to feedlots in Shanghai; China. J. Hazard. Mater. 2012, 235–236, 178–185. [Google Scholar] [CrossRef] [PubMed]
- Wang, R.; Chen, M.; Feng, F.; Zhang, J.; Sui, Q.; Tong, J.; Wei, Y.; Wei, D. Effects of chlortetracycline and copper on tetracyclines and copper resistance genes and microbial community during swine manure anaerobic digestion. Bioresour. Technol. 2017, 238, 57–69. [Google Scholar] [CrossRef]
- Grimsey, E.M.; Weston, N.; Ricci, V.; Stone, J.W.; Piddock, L.J. Over-expression of RamA, which regulates production of the MDR efflux pump AcrAB-TolC, increases mutation rate and influences drug-resistance phenotype. Antimicrob. Agents Chemo-Ther. 2020, 64, e02460-19. [Google Scholar]
- Pal, C.; Asiani, K.; Arya, S.; Rensing, C.; Stekel, D.J.; Larsson, D.J.; Hobman, J.L. Metal resistance and its association with antibiotic resistance. Adv. Microb. Physiol. 2017, 70, 261–313. [Google Scholar] [CrossRef] [PubMed]
- Amsalu, A.; Sapula, S.A.; Lopes, M.D.B.; Hart, B.J.; Nguyen, A.H.; Drigo, B.; Turnidge, J.; Leong, L.E.; Venter, H. Efflux pump-driven antibiotic and biocide cross-resistance in Pseudomonas aeruginosa isolated from different ecological niches: A case study in the development of multidrug resistance in environmental hotspots. Microorganisms 2020, 8, 1647. [Google Scholar] [CrossRef]
- Kaur, U.J.; Chopra, A.; Preet, S.; Raj, K.; Kondepudi, K.K.; Gupta, V.; Rishi, P. Potential of 1-(1-napthylmethyl)-piperazine, an efflux pump inhibitor against cadmium-induced multidrug resistance in Salmonella enterica serovar Typhi as an adjunct to antibiotics. Braz. J. Microbiol. 2021, 52, 1303–1313. [Google Scholar] [CrossRef]
- Ivanov, M.E.; Fursova, N.K.; Potapov, V.D. Pseudomonas aeruginosa efflux pump superfamily (review of literature). Klin. Lab. Diagn. 2022, 67, 53–58. [Google Scholar] [CrossRef]
- Seiler, C.; Berendonk, T.U. Heavy metal driven co-selection of antibiotic resistance in soil and water bodies impacted by agriculture and aquaculture. Front. Microbiol. 2012, 3, 399. [Google Scholar] [CrossRef]
- Ding, J.; An, X.L.; Lassen, S.B.; Wang, H.T.; Zhu, D.; Ke, X. Heavy metal-induced co-selection of antibiotic resistance genes in the gut microbiota of collembolans. Sci. Total Environ. 2019, 683, 210–215. [Google Scholar] [CrossRef]
- Di Cesare, A.; Eckert, E.; Corno, G. Co-selection of antibiotic and heavy metal resistance in freshwater bacteria. J. Limnol. 2016, 75, 59–66. [Google Scholar] [CrossRef]
- Delmar, J.A.; Su, C.-C.; Yu, E.W. Heavy metal transport by the C us CFBA efflux system. Protein Sci. 2015, 24, 1720–1736. [Google Scholar] [CrossRef] [PubMed]
- Martins, M.; Couto, I.; Viveiros, M.; Amaral, L. Identification of efflux-mediated multi-drug resistance in bacterial clinical isolates by two simple methods. In Antibiotic Resistance Protocols; Humana Press: Totowa, NJ, USA, 2010; pp. 143–157. [Google Scholar] [CrossRef]
- Timková, I.; Lachká, M.; Kisková, J.; Maliničová, L.; Nosáľová, L.; Pristaš, P.; Sedláková-Kaduková, J. High frequency of antibiotic resistance in deep subsurface heterotrophic cultivable bacteria from the Rozália Gold Mine, Slovakia. Environ. Sci. Pollut. Res. 2020, 27, 44036–44044. [Google Scholar] [CrossRef] [PubMed]
- Lachka, M.; Soltisova, K.; Nosalova, L.; Timkova, I.; Pevna, V.; Willner, J.; Janakova, I.; Luptakova, A.; Sedlakova-Kadukova, J. Metal-containing landfills as a source of antibiotic resistance. Environ. Monit. Assess. 2023, 195, 262. [Google Scholar] [CrossRef]
- Aleem, A.; Isar, J.; Malik, A. Impact of long-term application of industrial wastewater on the emergence of resistance traits in Azotobacter chroococcum isolated from rhizosphere soil. Bioresour. Technol. 2003, 86, 7–13. [Google Scholar] [CrossRef]
- Nosáľová, L. Štúdium Baktérií Z Biogeochemického Cyklu Zlata. Master’s Thesis, UPJŠ, Košice, Slovakia, 2019. [Google Scholar]
- Zgurskaya, H.I. Introduction: Transporters, Porins, and Efflux Pumps. Chem. Rev. 2021, 121, 5095–5097. [Google Scholar] [CrossRef] [PubMed]
- Paixão, L.; Rodrigues, L.; Couto, I.; Martins, M.; Fernandes, P.; de Carvalho, C.C.; Monteiro, G.A.; Sansonetty, F.; Amaral, L.; Viveiros, M. Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. J. Biol. Eng. 2009, 3, 18. [Google Scholar] [CrossRef]
- Chakotiya, A.S.; Tanwar, A.; Narula, A.; Sharma, R.K. Zingiber officinale: Its antibacterial activity on Pseudomonas aeruginosa and mode of action evaluated by flow cytometry. Microb. Pathog. 2017, 107, 254–260. [Google Scholar] [CrossRef]
- Molyneux, P.M.; Kilvington, S.; Wakefield, M.J.; Prydal, J.I.; Bannister, N.P. Autofluorescence signatures of seven pathogens: Preliminary in vitro investigations of a potential diagnostic for Acanthamoeba keratitis. Cornea 2015, 34, 1588–1592. [Google Scholar] [CrossRef]
- Bălăşoiu, M.; Bălăşoiu, A.T.; Mănescu, R.; Avramescu, C.; Ionete, O. Pseudomonas aeruginosa resistance phenotypes and phenotypic highlighting methods. Curr. Health Sci. J. 2014, 40, 85–92. [Google Scholar] [CrossRef]
- Aires, J.R.; Köhler, T.; Nikaido, H.; Plésiat, P. Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob. Agents Chemother. 1999, 43, 2624–2628. [Google Scholar] [CrossRef] [PubMed]
- Shinde, V.; Thombre, R. Antibiotic resistance profiling of marine halophilic bacteria and haloarchaea. J. Appl. Pharm. Sci. 2016, 6, 132–137. [Google Scholar] [CrossRef]
Figure 1.
Characterisation of the studied ore sampling sites of industrial waste from Hodruša (surface landfill (H/L), deep underground sampling sites (H/U1, (H/U2)) and Košice (surface landfill (K/L)) and soil sampling site from Hodruša (H/soil)).
Figure 1.
Characterisation of the studied ore sampling sites of industrial waste from Hodruša (surface landfill (H/L), deep underground sampling sites (H/U1, (H/U2)) and Košice (surface landfill (K/L)) and soil sampling site from Hodruša (H/soil)).
Figure 2.
Evaluation of fluorescence of bacterial isolates after the addition of increasing concentrations of ethidium bromide to the medium.
Figure 2.
Evaluation of fluorescence of bacterial isolates after the addition of increasing concentrations of ethidium bromide to the medium.
Table 1.
Concentrations of tested antibiotics and metals at the sampling sites (AMP—ampicillin, CHLOR—chloramphenicol, TET—tetracycline, KAN—kanamycin.
Table 1.
Concentrations of tested antibiotics and metals at the sampling sites (AMP—ampicillin, CHLOR—chloramphenicol, TET—tetracycline, KAN—kanamycin.
| Antibiotic Concentration (μg/mL) | |||||||||||
| AMP | - | - | 5 | - | 10 | 20 | - | 50 | 100 | - | 1000 |
| CHLOR | 1 | 2 | 5 | - | 10 | 20 | - | 50 | 100 | - | - |
| TET | - | 2 | 5 | 8 | 10 | 20 | - | 50 | 100 | - | - |
| KAN | - | - | 5 | - | 10 | 20 | 30 | 50 | 100 | 200 | - |
| Metal Concentration (μg/mL) | |||||||||||
| Cu | 100 | 125 | 250 | 500 | - | 1000 | - | - | - | - | - |
| Zn | 100 | - | 250 | 500 | - | 1000 | - | - | - | - | - |
| Pb | 100 | - | - | - | - | 1000 | 1500 | 2000 | - | - | - |
| Ni | 100 | - | 300 | 500 | 750 | 1000 | - | - | - | - | - |
Table 2.
Concentrations of metals/antibiotics used as limits to evaluate bacterial resistance to listed metals/antibiotics.
Table 2.
Concentrations of metals/antibiotics used as limits to evaluate bacterial resistance to listed metals/antibiotics.
| Metal/Antibiotic | AMP | CHLO | TET | KAN | Zn | Cu | Ni | Pb |
|---|---|---|---|---|---|---|---|---|
| Concentration (μg/mL) | 50 | 10 | 10 | 30 | 125 | 300 | 250 | 1000 |
Table 3.
MIC (minimal inhibitory concentration) of antibiotics and metals at sampling sites (values considered evidence of antibiotic/metal resistance are in bold). The shaded cell indicates concentrations of antibiotic/metal exceeding the threshold for antibiotic/metal resistance determination, indicating tolerance of the bacterium to the specific metal/antibiotic. * bacterial taxon for Hodruša determined by Nosalova et al. [27].
Table 3.
MIC (minimal inhibitory concentration) of antibiotics and metals at sampling sites (values considered evidence of antibiotic/metal resistance are in bold). The shaded cell indicates concentrations of antibiotic/metal exceeding the threshold for antibiotic/metal resistance determination, indicating tolerance of the bacterium to the specific metal/antibiotic. * bacterial taxon for Hodruša determined by Nosalova et al. [27].
| Sampling Site | ATB MIC (ug/mL) | MIC metals (ug/mL) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Bacterial Taxon * | AMP | CHLOR | TET | KAN | Zn | Ni | Cu | Pb | ||
| Hodruša | H/L | Rhizobium radiobacter | 800 | 5 | 5 | 10 | 250 | 250 | 750 | 2000 |
| Brachybacterium spp. | 10 | 1 | 50 | 5 | 1000 | 500 | 750 | 2000 | ||
| Comamonas testosteroni | 200 | 20 | 2 | 200 | 1000 | 500 | 750 | 2000 | ||
| H/U2 | Acidovorax spp. | 400 | 50 | 5 | 20 | 500 | 500 | 500 | 1500 | |
| H/soil | Rhodococcus erythropolis | 50 | 20 | 10 | 200 | 1000 | 500 | 500 | 2000 | |
| H/U1 | Staphylococcus spp. | 100 | 2 | 2 | 5 | 100 | 250 | 500 | 1500 | |
| NRI-11 | 10 | 1 | 2 | 5 | 125 | 100 | 100 | 1000 | ||
| Košice | K/L | NRI-1 | 10 | 5 | 100 | 200 | 100 | 300 | 150 | 2000 |
| NRI-2 | 5 | 5 | 100 | 500 | 100 | 300 | 150 | 2000 | ||
| NRI-3 | 5 | 2 | 5 | 200 | 100 | 300 | 200 | 2000 | ||
| Microbacterium sp. | 5 | 2 | 10 | 200 | 100 | 300 | 200 | 2000 | ||
| NRI-4 | 10 | 5 | 100 | 200 | 100 | 300 | 200 | 2000 | ||
| NRI-5 | 5 | 2 | 2 | 100 | 50 | 200 | 150 | 1000 | ||
| NRI-6 | 5 | 2 | 2 | 500 | 50 | 300 | 150 | 1000 | ||
| NRI-7 | 5 | 2 | 2 | 30 | 25 | 100 | 100 | 1000 | ||
| NRI-8 | 10 | 2 | 2 | 200 | 100 | 300 | 200 | 2000 | ||
| NRI-9 | 10 | 2 | 2 | 200 | 100 | 300 | 300 | 2000 | ||
| NRI-10 | 5 | 2 | 2 | 200 | 100 | 300 | 300 | 2000 | ||
| NRI-12 | 5 | 2 | 2 | 30 | 50 | 300 | 150 | 1000 | ||
Table 4.
Overproduction of efflux pump and concentrations of ethidium bromide, which caused fluorescence. Multiresistance is evaluated by MAR index calculation (MAR index indicating the presence of multiresistance is in bold). The shaded cells indicate the mutual presence of the multiresistance and the overproduction of efflux pumps.
Table 4.
Overproduction of efflux pump and concentrations of ethidium bromide, which caused fluorescence. Multiresistance is evaluated by MAR index calculation (MAR index indicating the presence of multiresistance is in bold). The shaded cells indicate the mutual presence of the multiresistance and the overproduction of efflux pumps.
| Sampling Site | Bacterial Taxon | Overexpression of Efflux Pumps | Ethidium Bromide Concentration (mg/mL) | MAR Index | |
|---|---|---|---|---|---|
| Hodruša | H/L | Rhizobium radiobacter | √ | 1 | 0.50 |
| Brachybacterium spp. | X | 0.63 | |||
| Comamonas testosteroni | √ | 1 | 0.88 | ||
| H/U2 | Acidovorax spp. | √ | 2.5 | 0.75 | |
| H/soil | Rhodococcus erythropolis | √ | 2.5 | 0.88 | |
| H/U1 | Staphylococcus spp. | X | 0.38 | ||
| NRI-11 | √ | 1 | 0.00 | ||
| Košice | K/L | NRI-1 | X | 0.38 | |
| NRI-2 | X | 0.38 | |||
| NRI-3 | X | 0.25 | |||
| Microbacterium sp. | X | 0.25 | |||
| NRI-4 | X | 0.38 | |||
| NRI-5 | X | 0.13 | |||
| NRI-6 | X | 0.13 | |||
| NRI-7 | X | 0.00 | |||
| NRI-8 | X | 0.25 | |||
| NRI-9 | X | 0.38 | |||
| NRI-10 | X | 0.38 | |||
| NRI-12 | X | 0.00 | |||
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Sincak, M.; Šoltisová, K.; Luptakova, A.; Sedlakova-Kadukova, J. Overproduction of Efflux Pumps as a Mechanism of Metal and Antibiotic Cross-Resistance in the Natural Environment. Sustainability 2023, 15, 8767. https://doi.org/10.3390/su15118767
AMA Style
Sincak M, Šoltisová K, Luptakova A, Sedlakova-Kadukova J. Overproduction of Efflux Pumps as a Mechanism of Metal and Antibiotic Cross-Resistance in the Natural Environment. Sustainability. 2023; 15(11):8767. https://doi.org/10.3390/su15118767
Chicago/Turabian StyleSincak, Miroslava, Katarína Šoltisová, Alena Luptakova, and Jana Sedlakova-Kadukova. 2023. "Overproduction of Efflux Pumps as a Mechanism of Metal and Antibiotic Cross-Resistance in the Natural Environment" Sustainability 15, no. 11: 8767. https://doi.org/10.3390/su15118767
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