Cadmium (Cd²⁺) is one of the most toxic nonessential heavy metals and its contamination poses significant risks to human health and ecosystems. Chronic exposure to Cd²⁺can lead to severe health issues in humans, including kidney damage, bone fractures, and cancers (Jarup et al., 1998; Waalkes, 2000). Due to its classification as persistent, bioaccumulative, and toxic, Cd²⁺ has been identified as one of the 31 Priority Chemicals for action under the U.S. Environmental Protection Agency’s (EPA) National Partnership for Environmental Priorities (US EPA, 2024). Together with lead and mercury, Cd²⁺ is one of only three heavy metals designated by the U.S. Environmental Protection Agency as priority hazardous air pollutants for which emissions must be eliminated or considerably reduced in industrial processes such as coal- and oil-fired power generation (US EPA, 2024). In ecosystems, Cd²⁺disrupts soil microbial activity and plant growth, reducing agricultural productivity and biodiversity (McLaughlin and Singh, 1999). Current policies aim to limit Cd²⁺emissions through regulations on industrial discharges and the use of Cd²⁺-containing fertilizers (United Nations Environment Programme, 2010). Despite these efforts, continuous monitoring and stricter enforcement of regulations are necessary to protect human health and the environment from Cd²⁺exposure.

Techniques for the removal and immobilization of heavy metals in contaminated soils have included physical, chemical, and biological methods, as well as a mixture of multiple methods (Lee et al., 2009; Sánchez-Castro et al., 2023). Physical methods include encapsulation (Pandey et al., 2012) and electrokinetic removal (Hawal et al., 2023); however, encapsulation only contains the contaminants in the soil (Liu et al., 2018) and electrokinetic removal is expensive and difficult to implement on a large scale (Sun et al., 2023). Chemical processes include stabilization (Lee et al., 2009), but changes in soil pH could decrease the effectiveness of the treatment since the metal is not removed (Lombi et al., 2003). Soil properties such as pH and organic matter content play crucial roles in Cd²⁺mobility and bioavailability (Kabata-Pendias, 2010). A combination of biological methods including phytoremediation and bio-augmentation poses a green method of removal and are being promoted to mitigate Cd²⁺contamination and restore soil health (Salt et al., 1995; Clemens, 2006; Simmer and Schnoor, 2022). For instance, species of cadmium-resistant microbes have been shown to have high removal efficiencies and increase seed germination and growth rates of plants (Lata et al., 2021; Arce-Inga et al., 2022). For heavy metal alleviation, a sustainable alternative to mitigate Cd²⁺soil toxicity includes the use of beneficial microbes with heavy metal removal abilities. Cadmium-tolerant bacteria (CdtB) such as Enterobacter sp., Bacillus sp., sulfate-reducing bacteria, and plant growth-promoting rhizobacteria (Arce-Inga et al., 2022; Bravo and Braissant, 2022) have received more attention in recent years. Future research should focus on enhancing the efficiency of remediation techniques and developing sustainable agricultural practices to prevent Cd²⁺accumulation in soils. Moreover, it is important to identify diverse CdtB for use in green and cost-effective remediation of contaminated soils. Indeed, the application of metal-resistant bacteria in bioremediation presents promising opportunities for wastewater treatment, the restoration of contaminated soils, and bioprecipitation (Diels et al., 1995; Sreedevi et al., 2022; Nnaji et al., 2024; Abbas et al., 2025; Qattan, 2025).

Bacteria exposed to heavy metals like Cd²⁺often develop resistance through similar genetic pathways used for antibiotic resistance, including efflux pumps and resistance genes (Seiler and Berendonk, 2012; Bravo and Braissant, 2022). These shared mechanisms can lead to the co-selection of CdtB and antibiotic-resistant bacteria in environments contaminated with heavy metals (Baker-Austin et al., 2006). Studies have shown that CdtB strains frequently exhibit resistance to multiple antibiotics, indicating a co-selection between metal resistance and multidrug resistance (Stepanauskas et al., 2006). Furthermore, horizontal gene transfer facilitated by mobile genetic elements such as plasmids can disseminate both metal-resistance and antibiotic-resistance genes among bacterial populations (Baker-Austin et al., 2006; Seiler and Berendonk, 2012). This relationship between metal and antibiotic resistance underscores the need for integrated management strategies addressing pollutants. Effective regulation of heavy metal emissions and prudent use of antibiotics are crucial to mitigating the spread of resistant bacterial strains in the environment.

The microbial metal reduction reaction is energy-dependent, and detoxifying bacteria such as CdtB require nutrients that may be limited in soils depleted of organic matter (Wagner-Döbler, 2003). Soil properties, particularly carbon and nutrient availability, and pH, significantly influence the success of bacterial reduction and subsequent volatilization and immobilization during bioremediation (Wagner-Döbler, 2003; Cattani et al., 2009). It has been reported that organic matter addition significantly increased As, Hg and Cd volatilization and immobilization from investigated soils (Yang et al., 2007; Huang et al., 2012; Liu et al., 2019). Also, adding phosphorus compounds to manage Cd²⁺ and Pb²⁺-contaminated soils provides microorganisms with an essential nutrient, thereby enhancing microbial activity in these soils (Park et al., 2011). Given that microbial metal reduction can be utilized to remove heavy metals from contaminated soils by bio-augmentation and nutrient supplementation, it is essential to identify nutrients such as carbon substrates that enhance microbial growth when cultured in the laboratory.

The average concentration of Cd²⁺ in uncontaminated soil worldwide is 0.36 mg/kg, but it can range from 0.01 to 1 mg/kg (Kubier et al., 2019). Depending on the type of pollution, soil Cd²⁺ concentrations can be up to 344 mg/kg (by leaching of solid waste) and 74 mg/kg (by atmospheric deposition) in contaminated soils (Voglar and Lestan, 2010; Bi et al., 2006). In this study, we characterized bacterial strains isolated from Cd²⁺ spiked soil (2,043 mg/kg) of about one order of magnitude compared to average prevailing concentration levels in highly contaminated soils yielding CdtB species. Given the reported higher frequencies of antibiotic resistance in bacteria within metal-contaminated ecosystems (McArthur and Tuckfield, 2000; Stepanauskas et al., 2006) it is crucial to monitor the selection for antibiotic resistance in bacteria with bioremediation potential, as this has significant implications for human health. Forsberg et al. (2012) found evidence of the lateral transfer of multidrug-resistant genes from soil bacteria to human pathogens, highlighting the importance of monitoring antibiotic-resistance in bacteria.

Antibiotic susceptibility testing has been done routinely using several methods such as the measurement of inhibition zones in agar diffusion tests (e.g., Kirby-Bauer), turbidity-based measurements, and the counting of colony-forming units after serial dilution (Jorgensen and Ferraro, 2009) or determination of the minimal inhibitory concentration (MIC) in serial dilutions as recommended by the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute [CLSI], 2023). These conventional methods rely on static acquisition of single data points or regular-interval growth determinations (off-line susceptibility testing) and overlook the antibiotic-specific dynamic growth profiles of target microorganisms. Theophel et al. (2014) demonstrated that different microorganisms exhibit varying susceptibility to different antibiotics at different growth stages and times. In this study, we utilized on-line analysis of antibiotic susceptibility testing to provide robust information about CdtB growth kinetics with high temporal resolution in the presence of varying antibiotic concentrations in 96-well Sensititre microplates. Moreover, the on-line susceptibility analysis integrates simultaneous cultivation and assessment of antibiotic impact on bacterial growth dynamics. By combining these approaches with carbon substrate utilization profiles (BIOLOG^® EcoPlate), a better understanding of CdtB growth kinetics and metabolic potential could be achieved.

The primary objectives of this study were to identify and characterize CdtB species inhabiting elevated Cd²⁺amended soil, as well as to evaluate their antibiotic resistance and carbon utilization profiles, in order to enhance the efficiency of Cd²⁺remediation in contaminated soils. In addition, we aimed at using the Comprehensive Antibiotic Resistance Database Resistance Gene Identifier (CARD-RGI) tool and Python applications to predict the antibiotic resistance genes (ARG) in isolated CdtB species. The findings of this study hold significant implications for the use of novel microorganisms in immobilization or detoxification of Cd²⁺in polluted soils and for the potential augmentation of the remediation process through biostimulation with optimal carbon substrates.

In this study, the bacterial species B. mobilis and C. campinensis were isolated from cadmium-amended soil with Cd²⁺ levels approximately one order of magnitude higher than those typically found in soils highly contaminated with Cd²⁺. This categorizes them as Cd²⁺ tolerant bacterial species, indicating their remarkable ability to survive in high Cd²⁺ concentrations.

C. campinensis (basionym: R. campinensis) was initially isolated from various heavy metal-contaminated industrial biotopes in Campine, northeastern Belgium (Goris et al., 2001), and from a heavy metal-contaminated playground in Salgótarján, Hungary (Abbaszade et al., 2020), demonstrating its adaptation and resistance to environmental stresses. Abbaszade et al. (2020) documented several typical metal-resistant genes and gene clusters in C. campinensis strain S14E4C, including cadA (cadmium-translocating P-type ATPase), czcB (cobalt-zinc-cadmium efflux RND transporter, membrane fusion protein), czcR (cobalt-zinc-cadmium resistance protein), among others. The B. mobilis strain MCCC 1A05942 was isolated from sediment of the Indian Ocean (Liu et al., 2017) and a closely related strain B. mobilis CR3 strain isolated from Cr-polluted soil sample in Qinghai, China, can remove Cr(VI) mainly through bio-reduction by reductase (Ye et al., 2024). Various reductase enzymes are involved in heavy metal reduction (Ojha et al., 2023) and APS reductase was shown to be efficient in immobilizing soil Cd²⁺ in cadmium-contaminated paddy soils (Liu et al., 2024). The identification and characterization of these Cd²⁺-tolerant bacterial strains highlight their potential use in the remediation of heavy metal-contaminated environments. Their ability to thrive in extreme conditions suggests they could be pivotal in developing biotechnological applications for the detoxification of cadmium-polluted soils. Additionally, understanding the genetic mechanisms underlying their resistance offers valuable insights for engineering more robust microbial strains for environmental clean-up strategies.

In recent years, a few microorganisms with the potential to absorb Cd²⁺ with varying efficiencies and limitations have been documented. For instance, Pseudomonas aeruginosa has been highlighted as a versatile microbe and a suitable biosorbent for the removal of Cd²⁺. It also promotes plant growth and enhances heavy metal accumulation by plants (Chellaiah, 2018). Additionally, Bacillus cereus, when associated with lawn plants, showed approximately 33% removal efficiency of Cd²⁺ from contaminated soil (Zhou et al., 2024). Rhizobium leguminosarum forms a symbiotic relationship with legumes, reducing Cd²⁺ toxicity in plants and potentially enhancing Cd²⁺ uptake and sequestration (Jach et al., 2022). Klebsiella planticola strain was shown to convert S₂O₃ into H₂S and precipitates Cd ions as insoluble sulfides (Kour et al., 2021). Additionally, in agricultural applications, Bacillus subtilis has been shown to promote plant growth and Cd²⁺ uptake in host plants, assisting phytoremediation processes by altering soil conditions to make Cd²⁺ more bioavailable (Li et al., 2022). Despite the promising strategy of bioremediation through microbial activity, the known versatile Cd²⁺resistant bacteria remain limited. Therefore, we recommend that the potential of Cd²⁺ tolerant bacterial strains B. mobilis and C. campinensis in this study should be explored for their effectiveness in Cd²⁺ bioremediation, potentially contributing to innovative solutions for soil contamination.

It is not surprising that these Cd-tolerant species utilized limited substrates needed for growth in extreme environments and might devote energy to producing resistance determinants allowing them to live in extreme environments. The results showed that both bacterial strains can metabolize a low variety of carbon substrates with different efficiencies, reflecting their ability to thrive in environments with limited nutrient availability. Such metabolic flexibility is critical for bacteria inhabiting heavy metal-contaminated soils, where nutrient sources may be limited or irregular. B. mobilis exhibited high metabolic activity when exposed to substrates like pyruvic acid methyl ester (carbohydrate), D-malic acid, Itaconic acid, D-Galactonic acid γ-Lactone (carboxylic and acetic acids) and phenylethylamine (amine/amides), suggesting that B. mobilis can efficiently utilize for instance both simple carbohydrates (itaconic acid) and more complex carbon substrates (Phenylethylamine, D-Galactonic acid γ-Lactone). The significantly utilized substrates for C. campinensis were complex polymers (Tween-40 and Tween-80), an amino acid (L. Asparagine), and a simple carbohydrate (pyruvic acid methyl ester).

Microbial metabolism of specific carbon substrates plays a critical role in shaping community responses to heavy metal stress, as these substrates can influence both metal mobility and microbial tolerance mechanisms. Organic acids, amino acids, and surfactants have been linked to enhanced microbial activity, exopolysaccharide production, and increased solubility or uptake of metals in contaminated soils (Skorokhodova et al., 2021; Kenarova and Boteva, 2015; Memarian and Ramamurthy, 2013; Cheng et al., 2017; Harati et al., 2023). In this study, we found that B. mobilis and C. campinensis disproportionately utilized several carbon substrates under Cd²⁺ stress, and these utilization patterns are consistent with mechanisms previously linked to microbial remediation of heavy metals. For example, pyruvic acid and D-malic acid, which were among the most highly utilized substrates in our assays, are known to enhance microbial respiration and biomass production, as well as stimulate exopolysaccharide production that can immobilize heavy metals (Skorokhodova et al., 2021; Si et al., 2022; Adedayo et al., 2023; Lou et al., 2023). Similarly, itaconic acid and D-galactonic acid-γ-lactone, also strongly metabolized in our study, have been shown to promote the growth of metal-binding microbes and to support microbial resistance to heavy metals (Krishnamoorthy et al., 2021; Kenarova and Boteva, 2015; Martínez-Toledo et al., 2021). Our data further revealed that phenylethylamine and L-asparagine supported the growth of metal-tolerant taxa, in line with earlier reports connecting these substrates to contaminant degradation pathways and reductions in bioavailable Zn (Koner et al., 2022; Ghorbanzadeh et al., 2022). Finally, we observed notable utilization of surfactants such as Tween-40 and Tween-80, which studies have shown their role in enhancing the solubility and microbial uptake of polycyclic aromatic hydrocarbons and metals, including Cd²⁺ and Pb²⁺ (Dhenain et al., 2006; Lima et al., 2011; Memarian and Ramamurthy, 2013; Cheng et al., 2017; Harati et al., 2023). Collectively, these findings demonstrate that the substrate preferences identified in our isolates align with known biostimulation mechanisms and highlight their potential for enhancing Cd²⁺ uptake and reduction in contaminated soils.

Bioaugmentation and biostimulation are well-established strategies for accelerating the on-site degradation of organic pollutants and facilitating metal reduction. Introducing specific carbon substrates such as organic acids or surfactants can be crucial in promoting the activity of native, metal-tolerant microorganisms, among which particular species may enhance the rate and efficiency of bioremediation. In this context, the six carbon substrates identified as highly utilized by B. mobilis and the four substrates identified for C. campinensis represent promising carbon substrates for further investigation. The above findings provide a foundational characterization of the Cd²⁺-tolerance and metabolic capacity of B. mobilis and C. campinensis for potential targeted biostimulation to boost in-situ bioremediation processes for heavy metals at contaminated sites. Although we did not evaluate their direct effects on plants under Cd²⁺ stress, evidence from related Bacillus and Cupriavidus strains suggests potential applicability in rhizosphere colonization, cadmium immobilization, and mitigation of metal-induced phytotoxicity (Chen et al., 2008; Rajkumar et al., 2010; Ma et al., 2016; Nayak et al., 2018; Yang et al., 2024). Future studies should specifically assess the plant-growth–promoting and stress-alleviating capacities of these isolates in cadmium-contaminated soils.

The dynamic effects of antibiotics on B. mobilis and C. campinensis growth patterns, as observed in this study, provide critical insights into how bacterial isolates from cadmium-amended soils respond to diverse antibiotic pressures. These results reveal significant differences in growth phases and rates, reflecting varying degrees of susceptibility and adaptive responses. Heavy metals like Cd²⁺are known to co-select for antibiotic resistance, as resistance genes often co-localize with metal resistance determinants on mobile genetic elements such as plasmids and transposons (Pal et al., 2015). This co-selection could explain the elevated baseline resistance observed in B. mobilis and C. campinensis, especially to antibiotics with overlapping resistance mechanisms, such as beta-lactams and aminoglycosides.

For C. campinensis, most tested antibiotics, including imipenem, tigecycline, tetracycline, gentamicin, levofloxacin, and ceftriaxone, were inhibitory at all concentrations. However, notable exceptions, such as the recovery of growth at low ceftriaxone concentrations (0.5 μg/mL), suggest concentration-dependent adaptation mechanisms. The extended lag phase observed at higher antibiotic concentrations, such as those of piperacillin and doripenem, likely represents a survival strategy allowing cells to endure sublethal antibiotic stress before resuming growth. Such lag phase extensions may involve upregulation of efflux pumps, stress response pathways, or the activation of antibiotic resistance genes, as previously described in resistant bacteria (Lambert, 2002; Baker-Austin et al., 2006; Martinez, 2009).

In contrast, B. mobilis exhibited greater resilience, with nearly 50% of tested antibiotics showing limited activity across all concentrations. This limited susceptibility could be attributed to intrinsic resistance mechanisms or the physiological robustness of B. mobilis under stress conditions. Notably, antibiotics such as erythromycin, clindamycin, daptomycin, ampicillin, and vancomycin were ineffective, with growth dynamics comparable to antibiotic-free controls. These observations are consistent with reports that Gram-positive bacteria, particularly environmental isolates, often possess structural barriers, such as thick peptidoglycan layers, and constitutive efflux pumps that confer broad-spectrum resistance (Baker-Austin et al., 2006; Martinez, 2009). The distinct growth patterns observed for levofloxacin, nitrofurantoin, ciprofloxacin, and streptomycin highlight the complexity of antibiotic concentration-dependent effects. For instance, B. mobilis exposed to levofloxacin exhibited an extended lag phase of up to 48 h at 1 μg mL^-1, followed by gradual growth, whereas above 2 μg mL^-1 there was complete inhibitory effect. This prolonged lag phase may indicate the activation of DNA repair systems, as levofloxacin targets bacterial DNA gyrase (Table 2), inducing double-strand breaks that necessitate extensive repair before replication can resume (Hooper and Jacoby, 2015). Similarly, the delayed growth of cells exposed to streptomycin underscores the potential for ribosomal protein modification or aminoglycoside inactivation enzymes to mitigate translational inhibition (Davies and Davies, 2010).

Our in silico analyses revealed comprehensive multidrug resistance (MDR) profiles in both strains, complementing their high Cd²⁺-tolerance; a hallmark of C. campinensis noted in other strains tolerating Cd levels up to ∼19 mM (Abbaszade et al., 2020). B. mobilis exhibited a broad ARG burden, including β-lactamases (blaZ, mecA), fluoroquinolone targets (gyrA, parC), macrolide-clindamycin methylases (ermB, ermC), and tetracycline efflux pumps (tetK, tetL). Such a profile reflects its environmental adaptability and may point to horizontal gene transfer from commensals or pathogens (Baker-Austin et al., 2006). The presence of mprF highlights its capacity to resist lipopeptide antibiotics like daptomycin (Thitiananpakorn et al., 2020), underlining the need for caution when considering its use in bioremediation.

Similarly, C. campinensis displayed a clinically alarming resistome profile, defined by strict to perfect matches to blaKPC-2, blaNDM-1, blaVIM-2, and blaOXA-48. These enzymes confer resistance to all tested carbapenems and are recognized as high-risk carbapenemase globally (Jean et al., 2022). Class A–D β-lactamases were well-represented, aligning with previously observed intrinsic resistome signatures in Cupriavidus. The detection of RND family efflux pumps -key mediators of broad-spectrum resistance-further underscores their potential clinical threat (Anes et al., 2015).

A particularly significant finding of this study is the persistence of growth effects well beyond the standard 24-h incubation period used in most clinical and environmental susceptibility testing protocols (CLSI, 2023). For example, in C. campinensis, antibiotics such as ceftriaxone, piperacillin, and ampicillin exhibited delayed inhibitory effects or adaptive growth after extended lag phases of 36–50 h. Similarly, in B. mobilis, lag phases of up to 48 h for ciprofloxacin and 30 h for nitrofurantoin reveal the potential for delayed bacterial adaptation to antibiotic stress. The lack of complete inhibition at higher concentrations highlights the potential for resistance mechanisms to confer partial protection, even under substantial antibiotic stress. These observations suggest that traditional susceptibility testing methods, which typically assess growth inhibition within 24 h, may overlook critical adaptive responses and underestimate the resilience of certain bacterial isolates. Extending susceptibility testing beyond the standard 24-h timeframe is crucial for understanding the full spectrum of bacterial responses to antibiotics (Theophel et al., 2014). Delayed growth recovery, as observed in this study, could have significant implications for treatment efficacy and the emergence of resistance. For instance, patients receiving antibiotics may initially appear to respond to treatment, but delayed bacterial recovery could lead to treatment failure or relapse. Additionally, sublethal antibiotic exposure during prolonged lag phases might facilitate the development of tolerance or resistance through selection of adaptive mutants or the activation of stress-induced resistance mechanisms (Theophel et al., 2014; Drawz and Bonomo, 2010). Ecologically, in contaminated ecosystems such as cadmium-amended soils, bacterial isolates are likely subjected to prolonged exposure to sublethal antibiotic concentrations due to environmental persistence of antibiotics and heavy metal co-selection pressures. The extended lag phases observed in this study could represent an ecologically relevant adaptation, allowing bacteria to survive fluctuating antibiotic concentrations. This highlights the need for susceptibility testing protocols that reflect environmental conditions more accurately, particularly in ecosystems where co-selection for antibiotic and metal resistance is likely (Baker-Austin et al., 2006; Pal et al., 2015).

Our findings emphasize the importance of dynamic, time-resolved assessments of bacterial growth in both clinical and environmental contexts. Automated OD measurements, as implemented in this study, provide a valuable tool for capturing differential growth dynamics and resistance profiles of B. mobilis and C. campinensis over extended time periods, offering a more comprehensive understanding of bacterial responses to antibiotics. Future studies should explore the molecular mechanisms underlying extended lag phases and delayed growth recovery, particularly in relation to heavy metal stress and resistance gene expression.

This study identified B. mobilis and C. campinensis as multidrug resistant and cadmium-tolerant bacteria with potential for bioremediation of heavy metal-contaminated soils. Both strains exhibited limited substrate utilization profiles, indicative of metabolic adaptations for survival in nutrient-poor, metal-stressed environments. Their ability to metabolize key substrates, such as organic acids and surfactants, supports their role in enhancing heavy metal bioavailability and mobilization in contaminated soils. Antibiotic susceptibility testing revealed delayed growth responses beyond the standard 24-h timeframe, highlighting the limitations of traditional testing protocols in capturing adaptive resistance mechanisms. We summarize predicted resistance genes in silico from available genomic data entered in CARD-RGI. Using a higher ARG-target coverage cutoff (>95%) predicted presence of seven and six perfect hits in B. mobilis and C. campinensis respectively. C. campinensis exhibited critical resistance to all tested carbapenems revealing a concerning carbapenem-resistant Enterobacteriaceae (CRE) profile. These findings underscore the ecological relevance of extended testing periods, particularly in environments where co-selection pressures from heavy metals and antibiotics persist. Also, the observed co-selection of resistance to both Cd²⁺and antibiotics further underscores the ecological complexity of contaminated environments, where heavy metal pressures and residual antibiotic concentrations may synergistically drive resistance evolution. Our study demonstrates that B. mobilis and C. campinensis isolated from cadmium-spiked soils exhibit both functional diversity in carbon metabolism and multidrug resistance traits, underscoring their ability to adapt and persist under high Cd²⁺ stress. These findings highlight the potential of these strains as candidates for application in bioremediation of heavy metal–contaminated environments, where metabolic versatility and resistance determinants are critical for survival and ecological function. While additional studies could further elucidate the genetic pathways underlying these traits, the present work provides clear evidence of their ecological resilience and biotechnological promise.