Prospecting the biodegradation of ciprofloxacin by Stutzerimonas stutzeri R2 and Exiguobacterium indicum strain R4 isolated from pharmaceutical wastewater

As the global population grows and industrialization and urban development continue, there is a rising demand for an improved quality of life. Various industries have emerged to meet human needs, yet their operations often generate wastewater, bringing about significant challenges (Rashid et al. 2021; Bhat et al. 2022; Othmani et al. 2022). This worldwide issue encompasses various industrial effluents that pose severe health risks, such as cancer, reproductive disorders, organ failure, and antibiotic resistance. Some of the key contributors to this problem include effluents from fertilizer, food and dairy processing, petrochemical, textile, and pharmaceutical industries (Rashid et al. 2020; Rashid et al. 2021). Among pharmaceutical compounds, antibiotics are important pharmaceutical compounds widely used in human and veterinary medicine (Baralla et al. 2021). Regardless of their benefits, antibiotics constitute an important class of emerging micropollutants of concern, because of their ecotoxicity and destructive biological effects (Miran et al. 2018; Thelusmond et al. 2018; Holanda et al. 2019). Since the preponderance of antibiotics is not fully metabolized by humans and animals, a substantial amount of administered antibiotics is released into the environment via anthropogenic waste (Feng et al. 2019). Besides, pharmaceutical and hospital wastewater also contain a significant concentration of toxic refractory compounds that persist in the environment for a long period (Leng et al. 2016; Nguyen et al. 2018). Recently, antibiotic pollution has garnered significant attention due to the presence of antibiotics in the environment, which exerts pressure favoring the survival of resistant bacteria. These bacteria pose a major public health concern due to the increased occurrence of associated infections (Singh et al. 2017; Manage 2018; Lalitha et al. 2019; Baralla et al. 2021; Mutuku et al. 2022). According to the Global Water Research Coalition's (GWRC) list of pharmaceuticals, ciprofloxacin (CIP), a broad-spectrum fluoroquinolone antibiotic that inhibits the key enzymes involved in DNA replication, including topoisomerase IV and DNA gyrase, was prioritized due to its potential threats to the water cycle and aquaculture. About 70% of the expelled CIP remains unmetabolized when released into the environment, and it has a half-life of up to 3,466 days in soil (Cycon et al. 2019; Feng et al. 2019; Cai et al. 2022). Hence, it has been selected as the model antibiotic for this study.

Ciprofloxacin is commonly found in the environment with concentrations ranging from 5.3 to 119.8 μg/kg in agricultural soil, 45.49 mg/kg in manure, 426 mg/kg in sewage sludge, 6.5 mg/L in freshwater ecosystem, and 6.5–31 mg/L in pharmaceutical wastewater (Nguyen et al. 2018; Feng et al. 2019). The distinctive physicochemical features, electronegativity, and C–F bond stability of fluorine enhance the antibacterial potential of CIP and transform it into a highly recalcitrant compound (Feng et al. 2019).

Ciprofloxacin can be removed by employing various physiochemical processes such as photodegradation, electrochemical oxidation, and advanced oxidation (Feng et al. 2019). However, these approaches are costly and sometimes ineffective, thus limiting their large-scale application. Furthermore, inefficient CIP degradation can produce intermediates with a toxicity similar to or higher than the parent molecule, resulting in secondary pollution and harmful consequences (Feng et al. 2019).

Using microorganisms for the remediation of CIP is an alternate method. Bioremediation, a process that utilizes microbes, can effectively break down hazardous substances into less toxic or nontoxic substances. Compared to other technologies, this method is generally 60–70% more cost-effective (Singh et al. 2017; Sadaf et al. 2022). The bacterial strains Thermus thermophiles and Bradyhizobium sp.GLC_01 have been reported for their use in bioremediation of CIP (Nguyen et al. 2018; Pan et al. 2018). There is still very limited information available about the biodegradation of CIP by individual bacterial strains or consortiums of bacterial strains. Hence, developing processes for complete and faster degradation of CIP is a critical need.

This study aims to isolate CIP-degrading bacteria from pharmaceutical wastewater. The strain was acquired by enrichment and isolation procedures, and their genotype were determined using 16S rRNA gene-based sequencing. After 16S rRNA gene-based sequencing the strains were identified as Stutzerimonas stutzeri R2 and Exiguobacterium indicum strain R4 members of the genus Stutzerimonas, formerly known as Pseudomonas stutzeri, gram-negative and rod-shaped, exhibit aerobic, chemoorganotrophic traits without spore formation. They play a vital role in biodegrading complex compounds across various environments (Lalucat et al. 2022; Mulet et al. 2023). Members of the genus Exiguobacterium, gram-positive and motile, can adapt aerobically or anaerobically based on oxygen availability. Isolated from diverse environments like hot springs, wetlands, and river water, these bacteria are known for their role in biodegrading complex compounds (Pandey 2020). The findings of this study revealed that both strains have the ability to degrade CIP. These results can contribute to eliminating antibiotics from the environment and have the potential to be utilized in designing engineering processes aimed at removing CIP from soil and water.

The untreated wastewater samples contaminated with ciprofloxacin were taken from local pharmaceutical industries of Islamabad, Pakistan, and carried to Applied and Environmental Microbiology lab at Quaid-i-Azam University Islamabad on the same day. The rationale for selecting pharmaceutical wastewater, rich in ciprofloxacin, is because bacteria in this environment might have evolved to degrade CIP due to constant exposure. Additionally, contaminated soils, water bodies receiving pharmaceutical waste, or ecosystems contaminated with CIP are valuable sources to isolate ciprofloxacin-degrading bacteria. Bacterial strains were isolated using an enrichment technique, selectively promoting the growth of ciprofloxacin-degrading bacteria in the presence of ciprofloxacin within the growth media, while inhibiting the growth of other microorganisms. After isolation, the R2 and R4 bacterial strains were investigated for degradation of ciprofloxacin. The degradation of the CIP was analyzed by UHPLC and spectrophotometry. The antibacterial potential of intermediates formed after the degradation of CIP by R2 and R4 was also evaluated.

To isolate CIP-degrading bacteria, an enrichment assay was performed with some modifications to a previously described method (Feng et al. 2019). Pharmaceutical wastewater samples, used to enrich CIP-degrading bacterial community, were collected from various local pharmaceutical industries of Islamabad, Pakistan. All these samples were mixed, and 1 mL of mixed samples was incubated at 37 °C into 100 mL Erlenmeyer flask containing 50 mL of M9 medium (Na₂HPO₄.7H₂O (1.28 g/L), KH₂PO₄ (0.3 g/L), NaCl (0.05 g/L), and NH₄Cl (0.1 g/L) supplemented with yeast extract as additional C source and ciprofloxacin hydrochloride (98% purity). At three day intervals, this culture was transferred to the fresh M9 medium. The progressive acclimation procedure involved gradually reducing the additional C source and increasing the concentration of CIP, from 1 to 0.25 g/L and from 50 to 100 mg/mL, respectively. Following this process, the culture was 10-fold serially diluted in normal saline and plated on M9 agar containing 100 mg/L CIP. After incubation, bacterial colonies with varying morphologies from the serially diluted plates (ranging from 10⁻⁴ to 10⁻⁷) were isolated and preserved in 40% glycerol for further studies.

The Invitrogen kit by Thermo Fisher Scientific was used to extract the genomic DNA bacterial isolates R2 and R4 as per the manufacturer's instructions. Several other extraction protocols, such as phenol–chloroform extraction, CTAB-based methods, and commercial kits from different manufacturers, are available and have been employed in various studies. This specific kit was chosen due to its documented effectiveness in extracting genomic DNA from bacterial cultures. The universal primers including 5′-AGAGTTTGATTCTGGCTCAG-3′ (27F, Forward primer) and 5′-GGTTACCTTGTTACGACTT-3′ (1492R, reverse primer) were used for the amplification of genes encoding 16S rRNA by PCR. Moreover, the PCR reaction mixtures consisting of dNTPs, Taq polymerase, buffer, forward and reverse primers, DNA template, and Milli-Q-water were used. After optimizing, the thermocycler was programmed to repeat 30 cycles with pre-heating carried out at 95 °C for 5 min, denaturation at 95 °C for 5 s, annealing at 55 °C for 30 s, extension at 72 °C for 90 s, and final polymerization at 72 °C for 10 min. Afterward, gel electrophoresis was used to validate the final PCR product. The gene sequencing was performed by Lab Genetix (Pakistan). Using the NCBI BLAST database, the query sequence was compared with other available sequences in the library to find its resembling sequences. The phylogenetic tree was constructed by the neighbor-joining method using MEGA11 software. The 16S rRNA sequence of the R2 and R4 strain were deposited in the GenBank under accession numbers OQ256706 and OQP423123, respectively.

The batch experiment was run to evaluate the biodegradation of CIP by test strains R2 and R4 under different experimental conditions described as follows. In experiment I, active bacterial cells were inoculated in the M9 medium containing CIP and yeast extract as additional C source (co-metabolism). In experiment II, active bacterial cells were inoculated in M9 medium containing CIP as a sole carbon source. Experiment III served as a control without bacterial cells to assess CIP degradation through abiotic factors. In all experiments, 100 mg/L of CIP was added, and the flasks were incubated at 37 °C with continuous agitation at 150 rpm. Samples were collected for analysis after 5 and 10 days to evaluate the biodegradation of CIP (Nguyen et al. 2018).

The degradation of the CIP was studied by ultra-high-performance liquid chromatography (UHPLC) and spectrophotometry. The selection of UHPLC and spectrophotometry was based on their accuracy, sensitivity in analyzing ciprofloxacin degradation, and available resources. The existence of alternative techniques such as mass spectrometry, HPLC coupled with mass spectrometry, or other chromatographic methods is acknowledged. Spectrometric determination of the remaining CIP was done by using a Lambda 25 UV/VIS spectrometer. A standard curve was generated using different concentrations of CIP (10–90 μg/mL) at 278 nm wavelength as λ_max to calculate CIP concentration.

The antibacterial potential of intermediates formed after the degradation of CIP by R4 was evaluated against Staphylococcus aureus and Escherichia coli on Muller Hinton agar by disc diffusion method (Singh et al. 2017).

In this study, statistical analyses including mean and standard deviation were computed using Microsoft Excel 2013. Statistix 8.1 software was used to analyze the significant difference in ciprofloxacin removal among three different treatments. This assessment was conducted through a two-way analysis of variance (two-way ANOVA), followed by the Tukey HSD post hoc test. Values were considered significant when p < 0.05.

S. stutzeri R2 and E. indicum R4 two of the seven bacterial strains, isolated from pharmaceutical wastewater by enrichment and acclimation method, which showed resistance against CIP. Acclimatization to a considerably high CIP concentration and a low proportion of secondary C sources may improve bacterial adaptability to antibiotic resistance as well as its potential to biodegrade pollutants. Furthermore, the adaptation of bacteria to high CIP concentration may cause considerable alterations in the diversity and structure of the microbial communities. Constant exposure to a rather high proportion of CIP might be the primary driving factor for the evolution and enzyme induction in CIP-degrading bacteria (Feng et al. 2019). Such biotransformation features might emerge and propagate throughout microbial communities when microbial progeny acquires these functional gene alleles from their parents. Moreover, favorable co-substrates, which are auxiliary compounds or substrates present in the environment, can enhance the biodegradation process of a specific target compound, thereby fostering greater diversity within the microbial community (Eliasson et al. 2023). After enrichment with CIP, the strains R2 and R4 were isolated with the ability to grow on the solid M9 medium containing 100 mg/L of CIP.

The genus Stutzerimonas, recently proposed within the Pseudomonadaceae family, includes species that were previously classified under the genus P. stutzeri, specifically those belonging to the P. stutzeri phylogenetic group (Gomila et al. 2022; Mulet et al. 2023). The phenotypic characteristics of genus Stutzerimonas include gram-negative, rod-shape, and non-spore-forming, aerobic, strictly oxidative, and chemoorganotroph. The phenotypic characteristics of the genus Stutzerimonas include being gram-negative, rod-shaped, non-spore-forming, aerobic, strictly oxidative, and chemoorganotrophic. Members of this genus have been isolated from a wide range of environments, including soil, plants, wastewater, marine water, sediments ranging from 11,000 meters in depth to intertidal shores, as well as from oil-contaminated sand, hydrothermal vents, and sludge (Lalucat et al. 2022). Members of the Stutzerimonas (formerly P. stutzeri) genus have been studied for their significant role in the biodegradation of various complex compounds, including agrochemicals (Xu et al. 2023), cyanide, phenol (Singh et al. 2018), benzo[a]pyrane (Kumari & Chandra 2023), as well as antibiotics like sulfamethoxazole and chloramphenicol (Yang et al. 2021).

Furthermore, the species of genus Exiguobacterium are a gram-positive, motile, and non-spore-forming. They can also be either aerobic or anaerobic, depending on the availability of oxygen and growth conditions. The members of this genus have been isolated from a wide range of environments, including hot springs, glaciers, rhizospheres, food processing plants, wetlands, river water, and plastic dumped soil (Chauhan et al. 2018; Maroof et al. 2022). The member of the genus Exiguobacterium have been investigated to play an important role in the biodegradation of a diverse range of complex compounds, viz., pesticides, heavy metals, 4-chloroindole, and triphenylmethane dye (Pandey 2020). Exiguobacterium undae strain DR14 and Exiguobacterium sibiricum strain DR11 have been reported to show promising biodegradation potential against polystyrene (Chauhan et al. 2018). Exiguobacterium Sp. strain LM-IK2 isolated from plastic dumped soil was found to degrade low-density polyethylene (Maroof et al. 2022). Furthermore, bacteria like Bradyrhizobium, Thermus thermophilus, and Geobacillus thermoleovorans are metabolically active microorganisms that are capable of degrading CIP originating from various sources. Meanwhile, S. stutzeri R2 and E. indicum R4 represent newly identified genera with the potential to efficiently degrade CIP. These strains, isolated in the current study, could be harnessed for use in diverse wastewater treatment systems, such as trickling filters, to remove the antibiotic CIP from wastewater.

To the best of our knowledge, this is the first report for the species of Stutzerimonas and Exiguobacterium sp. involved in CIP degradation and appears to be a promising candidate for the degradation of CIP. In the present study, the degradation efficiency of CIP by S. stutzeri R2 is higher than that of E. indicum R4. The notable difference in the degradation rates of CIP between S. stutzeri and E. indicum can be attributed to various factors. S. stutzeri demonstrated a higher degradation rate, possibly due to its possession of specialized enzymes. Moreover, more favorable growth conditions or more effective adaptation might have contributed to its superior performance compared to E. indicum. Further exploration into enzyme profiling and metabolic pathways would be beneficial in understanding the underlying reasons for the substantial disparity in CIP degradation rates between these two bacterial species. These findings align with previous research demonstrating that favorable growth conditions or more effective adaptation significantly influenced the degradation efficiencies of cefalexin by two other strains, Rhizobium sp. (CLX-2) and Klebsiella sp. (CLX-3) (Tian et al. 2023). However, it was also observed that the degradation rate of both the isolates, S. stutzeri R2 and E. indicum R4, is greater than previously reported values by other organisms. Bradyhizobium sp.GLC_01 degrades only 26.4% CIP at the concentration of 10 mg/L (Nguyen et al. 2018). After 11 days of incubation, freshwater microalgae Chlamydomonas mexicana removed only 13% of 2 mg/L CIP (Xiong et al. 2017). Thermus thermophiles, a thermophilic bacterial strain, degraded 55% of CIP (Pan et al. 2018).

The findings revealed that the biotransformation of CIP via co-metabolism was higher as compared to direct metabolism in which CIP was used as a sole carbon source. In this study, the addition of co-substrates boosted the growth of microorganisms that degrade pollutants, resulting in the rapid degradation of CIP. The additional substrates could have enabled microbial cells to mitigate CIP toxicity by providing sufficient nutrients. Consequently, this production of more biocatalysts capable of reacting with CIP could lead to faster degradation of CIP through energy-driven co-metabolism. These results are in accordance with those reported previously that showed almost complete biodegradation of CIP by the XG consortium upon supplementation of co-substrate (Feng et al. 2019).

The auxiliary organic feed not only assists the microbial cells in their growth, proliferation, and maintaining viability but also stimulates certain non-specific enzymes that help in the co-metabolic biodegradation of environmental contaminants. Organic compounds play a pivotal role by serving as vital nutrient sources, supplying carbon, energy, and metabolic substrates necessary for microbial growth, proliferation, and maintenance of cellular viability. Additionally, certain organic compounds can act as inducers, stimulating the production of enzymes not only crucial for their own breakdown but also aiding in the co-metabolic degradation of environmental pollutants. However, while these compounds generally support microbial functions, there exists the potential for adverse effects, including toxicity or inhibition at higher concentrations, alterations in microbial community dynamics, and the possibility of reducing the efficiency of contaminant degradation pathways through misdirected resource utilization (Luo et al. 2014; Feng et al. 2019). In the natural environment, CIP co-exists with a variety of rapidly biodegradable substrates, and therefore co-metabolic degradation potentially plays a key role in the bioremediation of pseudo-persistent pharmaceutical pollutants (Kiel & Engesser 2015; Murphy 2016).

In the current investigation, the addition of co-substrate promoted the growth of CIP-degrading bacteria, S. stutzeri R2 and E. indicum R4, leading to the rapid biotransformation of CIP. The co-substrate might have allowed the bacterial cells to substantially reduce the toxicity of CIP by providing sufficient nutrients, resulting in the production of biocatalysts that can potentially interact with CIP, hence triggering its biodegradation (energy-driven co-metabolism). Nevertheless, current research has established the practical significance of the requisite carbon sources as co-substrates for improved degradation of CIP by bacteria. Furthermore, the type of co-substrate utilized may be an important element in influencing CIP degradation efficiency (Maia et al. 2014; Pan et al. 2018).

Degraded CIP was tested for antimicrobial activity against E. coli and S. aureus. The antibacterial potency of CIP degradation products, formed after 5 and 10 days of incubation with S. stutzeri R2 and E. indicum R4, was found to be weaker than that of the CIP standard.

It was observed that an increase in the incubation time of CIP with S. stutzeri R2 and E. indicum R4 reduced the antibacterial potential of its degraded products. The decline in antibacterial activity may be attributed to the possibility that during biodegradation, ciprofloxacin undergoes chemical modifications or breaks down into metabolites that no longer possess the original antibiotic properties. These metabolites might exhibit altered chemical structures or modified functional groups, resulting in a diminished antibacterial efficacy. These outcomes are supported by a previous study that reported the decline in the antibacterial potential of CIP at an average of 26.3% by fungus Pleurotus ostreatus upon incubation of 14 days (Singh et al. 2017). These findings are also noteworthy because they indicate that treating CIP effluent with S. stutzeri R2 and E. indicum R4 may effectively decline its antibacterial properties, thus, potentially reducing the emergence of antibiotics resistance bacteria in the environment.

The presence of pharmaceutical pollutants in the environment has crucial impacts on the structure and function of the ecosystem and the emergence of antibiotic-resistant bacteria (Dąbrowska et al. 2018). The study concludes that the bacterial strains S. stutzeri R2 and E. indicum R4, which were isolated from pharmaceutical wastewater, exhibited considerable potential for degrading ciprofloxacin. It was observed that after 10 days of incubation, S. stutzeri R2 remove 77% of CIP, a higher degradation rate compared to the 68% demonstrated by E. indicum R4. Importantly, it was noticed that the biotransformation of CIP was found to be higher in co-metabolism compared to direct metabolism (using CIP as a sole carbon source). When CIP was used as the sole carbon source, the degradation of CIP was 35% for R2 and 28% for R4 after 10 days. Additionally, the products generated after degradation of CIP were having reduced antibacterial activity against test bacterial strains as detected from their zone of inhibition. It was noted that prolonging the incubation time of CIP with S. stutzeri R2 and E. indicum R4 led to a decrease in the antibacterial potency of its degraded products. The study's findings hold considerable importance. Therefore, it can be concluded that S. stutzeri R2 and E. indicum R4 are promising candidates for introducing into wastewater treatment systems like trickling filters to remove CIP from pharmaceutical and hospital wastewater before entering the natural environment. This proactive measure might mitigate the proliferation of antibiotic-resistant bacteria in natural environments.

The authors acknowledge the National Institute of Health Islamabad for providing UHPLC analyses facility.

Conceptualization: Q.A., S.A., and M.B.; Methodology: Q.A., S.A., and R.Z.; Formal analysis and investigation: Q.A., R.Z., and W.S.; Results analysis: Q.A., R.Z., G.M., and T.I.; Writing – original draft preparation: Q.A.; Writing – review and editing: Q.A., S.A., M.B., S.K., and W.S.; Funding acquisition S.A.; Resources: S.A., and M.B.; Supervision: S.A.

This study was funded by the Higher Education Commission of Pakistan. Q.A. was awarded the HEC Indigenous Scholarship granted by the Higher Education Commission of Pakistan.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.