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Article

Combating Environmental Antimicrobial Resistance Using Bacteriophage Cocktails Targeting β-Lactam-Resistant High-Risk Clones of Klebsiella pneumoniae and Escherichia coli in Wastewater: A Strategy for Treatment and Reuse

by
María D. Zapata-Montoya
†,
Lorena Salazar-Ospina
† and
Judy Natalia Jiménez
*
Research Group in Basic and Applied Microbiology (MICROBA), School of Microbiology, University of Antioquia, Medellín 050010, Colombia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Water 2025, 17(15), 2236; https://doi.org/10.3390/w17152236
Submission received: 29 June 2025 / Revised: 20 July 2025 / Accepted: 23 July 2025 / Published: 27 July 2025
(This article belongs to the Special Issue Evaluation of Microbiological Indicators for Water and Wastewater Treatment and Reuse)
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Abstract

Wastewater is a hotspot for the spread of antimicrobial resistance (AR); therefore, bacteriophages offer a promising biocontrol alternative to overcome the limitations of conventional disinfection. This study evaluated the efficacy of bacteriophages and cocktails for the biocontrol of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) (CG258 and ST307) and Escherichia coli producers of extended-spectrum β-lactamases (ESBL-Ec) (ST131) in simulated wastewater. A synthetic wastewater matrix was prepared in which bacterial viability and bacteriophage stability were assessed for 72 h. CR-Kp or ESBL-Ec strain were treated with individual bacteriophages or phage-cocktails (dosed in different ways) and bacterial loads were monitored for 54 h. The Klebsiella phages FKP3 and FKP14 eliminated 99% (−2.9 Log) of CR-Kp-CG258 at 54 h, and FKP10 reduced 99% (−2.15 Log) of the CR-Kp-ST307 strains. The Klebsiella phage-cocktail in a single dose reduced to 99.99% (−4.12 Log) of the CR-Kp-CG258 at 36 h. Coliphage FEC1 reduced to 2.12 Log (99%) of ESBL-Ec-blaCTX-M-G9, and FEC2 and FEC4 reduced approximately 1 Log (90%) of ESBL-Ec-blaCTX-M-G9 and blaCTX-M-G1. The coliphage cocktail increased the reduction up to 2.2 Logarithms. This study provides evidence supporting the use of bacteriophage cocktails for the control of resistant bacteria in wastewater, a sustainable intervention to mitigate the spread of AR and support water reuse safety.

Graphical Abstract

1. Introduction

The inappropriate use of antibiotics associated with various anthropogenic activities has led to an increase in the presence of antimicrobial-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotics in water, becoming a new class of emerging pollutants that have a great impact on aquatic ecosystems and human health [1]. In this context, wastewater treatment plants (WWTPs) are considered important sources of contamination and are beginning to be seen as reservoirs and places where the bacterial resistance phenomenon is especially intense, which favors the dissemination of these microorganisms to the environment. Some studies have documented the presence of carbapenem-resistant Klebsiella pneumoniae carrying the carbapenemase KPC (Klebsiella pneumoniae carbapenemase) and E. coli resistant to cephalosporins, producers of extended-spectrum β-lactamases (ESBL-Ec), in community domestic wastewater and WWTP effluents [2]. These β-lactam antibiotic-resistant enterobacteria are considered critical priority pathogens by the World Health Organization (WHO) due to their high number of infections, lack of therapeutic options, and high mortality rates [3].
The increase of these resistant bacteria in wastewater occurs mainly because conventional disinfection methods are not designed to specifically eliminate the ARB and ARGs present in wastewater [4]. However, some secondary treatments can favor the relative abundance of nutrients that provide a favorable environment for resistant bacteria and horizontal gene transfer [5]. On the other hand, although tertiary disinfection treatments (chlorination, UV radiation, and photocatalysis, among others) have been described to reduce ARB and ARG loads in wastewater, they are difficult to access due to their high cost and, in some cases, are complex to operate [5]. Therefore, exploring novel treatment strategies that are both cost-effective and environmentally sustainable and that align with the One Health framework is imperative to generate positive outcomes for both ecosystem integrity and health [4,5].
In this regard, bacteriophages have been proposed as an alternative to help mitigate this problem, and in recent years has increased interest in their use as selective bacterial biocontrol agents for secondary or tertiary wastewater treatment [6]. Bacteriophages are viruses that specifically infect bacteria, and they have been widely used in the context of health and industry. In particular, virulent phages are attractive biocontrol agents because they can recognize specific bacterial receptors, replicate using bacterial metabolism, and release progeny at the expense of lysis and bacterial death. In addition, they have activity against bacterial biofilms present in wastewater, can be used at low doses, and are self-limiting and conditioned to the presence of the bacteria they infect [7].
Considering the above and the high prevalence of resistant bacteria in wastewater and WWTP effluents, this study aimed to evaluate the efficacy of lytic bacteriophages for the elimination of resistant bacteria of high epidemiological importance, specifically carbapenem-resistant K. pneumoniae (CR-Kp) (CG258 and ST307 clones) and extended-spectrum β-lactamase (ESBL)-producing E. coli (ESBL-Ec) in simulated wastewater. This study presents bacteriophages as promising and sustainable biocontrol agents to mitigate the spread of antimicrobial resistance (AMR) in aquatic ecosystems, contributing to improved water quality for safe reuse and offering important public health benefits. Unlike previous studies, our work specifically addresses the biocontrol of multidrug-resistant bacteria in wastewater by targeting high-priority pathogens, including clinically relevant clones resistant to β-lactam antibiotic resistance profiles rarely evaluated in similar research. In addition, we optimized phage application protocols under controlled conditions, assessing sequential and repeated dosing strategies that enhanced phage persistence and biocontrol efficacy.

2. Materials and Methods

2.1. Isolation and Characterization of Bacteriophages

Bacteriophages were isolated from wastewater collected from a tertiary hospital and the effluent of a wastewater treatment plant (WWTP) in Medellín, Colombia. Phage isolation was performed using a diverse panel of host strains belonging to distinct clonal lineages. For Klebsiella pneumoniae, isolation targeted carbapenem-resistant strains (CR-Kp) from clonal group CG258 (ST512, ST258) and ST307. For Escherichia coli (coliphages), strains producing extended-spectrum β-lactamases (ESBL-Ec), representing multiple phylogroups and clones—including ST131, ST2, and others carrying the bla-CTX-M gene—were used as hosts. The isolation and characterization of phages were performed according to the protocol of Tellez et al. [8]. Briefly, the characterization included host range, efficiency of plating (EOP), bacterial kill curves, transmission electron microscopy (TEM) and determination of stability at different temperatures and pH conditions. Finally, the best bacteriophages were selected for the biocontrol assays according to their biological and structural characteristics.

2.2. Preparation of the Bacteriophage Cocktails

Two phage cocktails were formulated by combining the three specific phages for each bacterial model. FKP3, FKP10, and FKP14 were used against CR-Kp strains, whereas FEC1, FEC2, and FEC4 were used against ESBL-Ec strains. Each cocktail was prepared in SM buffer at a final concentration of 5 × 108 PFU/mL.

2.3. Selection of Bacteria for Biocontrol Assays

Three carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strains harboring blaKPC2 or blaKPC3, belonging to CG258 (ST512 and ST258) and ST307, were selected. Additionally, three extended-spectrum β-lactamase-producing E. coli (ESBL-Ec) strains carrying blaCTXM-G1 (n = 1) and blaCTXM-G9 (n = 2), associated with ST131 and phylogroups B2 and E, were included. Strains were obtained from the collection of the MICROBA research group and selected based on previous reports of prevalence in hospital and community wastewater effluents [2,9,10,11]. Identification and susceptibility testing were performed using the VITEK® 2 Compact system (BioMérieux, Marcyl’ Etoile, France). Resistance genes were detected using PCR, and sequenced. MLST and the Clermont method were used to determine sequence types and E. coli phylogroups [12,13]. Table 1 summarizes the characteristics of the bacteria used in the biocontrol assays.

2.4. Preparation of Synthetic Wastewater

Synthetic wastewater (SWW) was prepared as described by Serna-Galvis et al. [14] with modifications that reflect the typical components and concentrations found in community wastewater effluents. To minimize variability and ensure reproducibility, this study employed synthetic wastewater (SWW), which is a chemically defined formulation designed to simulate the key physicochemical parameters of municipal sewage. This standardizes experimental conditions, reduces biological and chemical variability, and allows better reproducibility between experimental replicates. The individual components were prepared separately, sterilized by 0.22 μm filtration (Minisart®, Sartorius AG, Göttingen, Lower Saxony, Germany), and mixed in ion-free water. The pH was adjusted to 7.3, and the final medium was autoclaved at 121 °C for 20 min [14]. The component concentrations are summarized in Table 2.

2.5. Bacterial Viability in Synthetic Wastewater

Prior to the biocontrol experiments with bacteriophages, bacterial viability assays were conducted independently for K. pneumoniae and E. coli strains to assess their survival under experimental conditions. These preliminary evaluations ensured the stability of the bacterial populations in the synthetic wastewater with restricted nutrient levels and under non-optimal growth temperature conditions. Bacterial viability assays were performed through quantitative plating on plate count agar (PCA) following Kauppinen et al. protocol with some modifications [15]. Cultures were grown in LB broth (BD, Difco, Sparks, MD, USA) supplemented with 2 mM CaCl2 (Biobasic, Markham, Ontario, Canada) and incubated at 35 ± 2 °C for 18–24 h under agitation at 120 rpm. A bacterial inoculum (~6 × 108 CFU/mL) was prepared from exponential-phase culture, centrifuged (4500 rpm, 10 min), washed twice, and resuspended in 1 mL of synthetic wastewater. This inoculum was added to 400 mL of synthetic wastewater to achieve a final concentration of 1 × 105 CFU/mL and incubated at 25 °C with agitation (120 rpm) for 72 h [15,16,17]. Samples (1 mL) were collected at 0, 3, 6, 18, 24, 48, 54, and 72 h. Each sample was mixed, serially diluted (1:10) in 0.9% NaCl, and plated on plate count agar (PCA)(Merck, Darmstadt, Germany) using the quantitative seeding method. The plates were incubated at 35 ± 2 °C for 18–24 h. Bacterial concentrations were expressed as CFU/mL [17,18]. All assays were performed in triplicate.

2.6. Stability of Bacteriophages in Synthetic Wastewater

The stability of bacteriophages over the 72 h experimental period was assessed by quantifying phage particles using the double-layer agar method. For each experiment, bacteriophages were added to the simulated wastewater until a final concentration of 1 × 108 PFU/mL. Bacteriophage stability assays were performed in triplicate in Erlenmeyer with 400 mL of synthetic wastewater and kept in agitation at 120 rpm for 72 h at 25 °C. During the assay, 1 mL of wastewater was collected at the beginning of the experiment (t0) and at hours 3, 6, 18, 24, 48, 54, and 72. Samples collected at each time were mixed, serially diluted (1:10) in SM buffer (100 mM, NaCl Merck Millipore; 50 mM Tris-HCl [pH 7.5]; 8 mM, MgSO4 Scharlau; 0.01% Oxoid gelatin), and seeded using the double-layer agar method. The cultures were incubated for 18–24 h at 35 ± 2 °C. At each time, the bacteriophage concentration (PFU/mL) in the synthetic wastewater was calculated [17,18].

2.7. Biocontrol Assays Using Bacteriophages in Synthetic Wastewater

K. pneumoniae and E. coli cultures were prepared as in the “Bacterial viability” and inoculated into synthetic wastewater until a final concentration of 1 × 105 CFU/mL. Bacteriophages or phage cocktails were then added to achieve 1 × 106 PFU/mL (MOI = 10) at the point when the bacterial density had stabilized (t0). The assays were performed in triplicate in 400 mL of synthetic wastewater, agitated at 120 rpm, and maintained at 25 °C for 54 h. Samples were collected at 0, 6, 30, 36, and 54 h. Bacteria were quantified on PCA (35 ± 2 °C, 18–24 h incubation) using the quantitative seeding method, and for individual-phage treatments, phage titers were determined by the double-layer agar technique [15,16,17]. CFU/mL and PFU/mL were calculated for each time point [19].

2.8. Biocontrol Assay Using Individual Bacteriophages

The phages FKP3 and FKP14 were applied to the CR-Kp-CG258 strains ST512 (KP58) and ST258 (KR06), respectively, whereas FKP10 targeted ST307 (KH45). For ESBL-Ec strains, FEC1 was used against HD67 (blaCTXM-G1, B2-phylogroup), FEC2 against HD184 (blaCTXM-G9, ST131 B2-phylogroup), and FEC4 against HD163 (blaCTXM-G1, E-phylogroup). Each phage was added once at t0, and the assays included (i) bacterial viability controls, (ii) phage stability controls, and (iii) individual phage treatments. Phage titers post-treatment was determined by the double-layer agar method, as previously described [15,16,17].

2.9. Biocontrol Assay Using Bacteriophage Cocktails

Bacteriophage cocktails were used to improve treatment performance and prevent the appearance of bacteria resistant to bacteriophages. The cocktails were used for the biocontrol of two strains of CR-Kp belonging to CG258 (ST512; KP58 and ST258; KR06); and one isolate of CR-Kp from ST307 (KH45). The phage-cocktail for E. coli was used in ESBL-Ec (blaCTXM-G1 and blaCTX-M-G9) belonging to ST131 and phylogroups B2 (HD184) and E (HD163). The bacteriophage cocktail was added once to the synthetic wastewater at t0. The assays included bacterial viability control, and treatment with the bacteriophage cocktail [15,16,17].

2.10. Biocontrol Assays Using Multiple Doses of the Cocktail

To optimize the performance of the bacteriophage cocktail in synthetic wastewater, a multi-dose administration strategy was evaluated. The assay included two CR-Kp strains KP58 (CG258-ST512) and KH45 (ST307) hosts of the FKP3 and FKP10 phages, respectively. For E. coli, an ESBL-producing strain (blaCTX-M-G9, ST131, B2-phylogroup) was used (HD163). The phage cocktail was administered thrice: at the start of the experiment (t0), at 6 h, and at 30 h. The assay design included bacterial viability control, and three-dose cocktail treatment [20].

2.11. Biocontrol Assays Using Individual and Sequential Administration of the Bacteriophages Composing the Cocktail

To compare the performance of the bacteriophage cocktail in synthetic wastewater, its components were individually and sequentially administered over time. For CR-Kp biocontrol assays targeting the CG258-ST512 strain (KP58), only FKP3 and FKP14 were used, and FKP10 was excluded due to its lack of individual lytic activity against this strain. FKP3 was administered at t0, followed by FKP14 at 30 h. For ESBL-Ec HD163 (bla-CTX-M-G1; B2-phylogroup), the three-phage, FEC1 (dosed at t0), FEC2 (at 6 h), and FEC4 (at 30 h) were added. Each experiment included bacterial viability control, and sequential individual phage administration. The dosing order was based on the biocontrol performance of individual phages and previous characterization data [8].

2.12. Evaluation of Bacteriophage Resistance

To determine the effect of biocontrol treatments with bacteriophages on bacteria, we described the phenotypic changes in colonies not eliminated by the phages. These colonies were considered possible bacteriophage-resistant bacterial subpopulations in wastewater. Colonies that exhibited changes in size and typical morphology were isolated on brain–heart infusion (BHI) agar through two consecutive streakings (Merck, Kenilworth, NJ, USA) and incubated for 18–24 h at 35 ± 2 °C to determine if the changes persisted across generations. Subsequently, the susceptibility of the obtained colonies to bacteriophages was evaluated using the spot test [15,17]. Finally, some of the phage-resistant bacteria were selected, seeded on chromogenic media (CROMID®CARBA, BioMérieux, Marcyl’ Etoile, France), and tested for antibiotic susceptibility using the automated Vitek 2 system. Susceptibility tests were performed on untreated and bacteriophage-susceptible strains.

2.13. Statistical Analysis

Quantitative variables are presented as mean and standard deviation. Reduction percentages are reported as per Mascarenhas et al. [19]. To compare the performance of each treatment, parametric and nonparametric tests were performed based on the results of the normality hypothesis evaluated using the Shapiro–Wilk test. Parametric tests included analysis of variance (ANOVA) or Student’s t-test, whereas nonparametric tests included the Kruskal–Wallis test or the Mann–Whitney U-test. Values of p < 0.05 were considered statistically significant. The data obtained were analyzed using RStudio v2024.12.1 + 563.

3. Results

3.1. Bacteriophages Selected After Isolation and Characterization

Six lytic bacteriophages were selected for biocontrol assays because they exhibited the most favorable biological and structural characteristics as detailed in previous studies [8]. Three of these were active against CR-Kp strains and were designated FKP3, FKP14, and FKP10, while the coliphages were active against ESBL-Ec strains and they were called FEC1, FEC2, and FEC4.
Two K. pneumoniae bacteriophages (FKP3 and FKP14) exhibited specificity for 56–60% of the GC258 strains, while FKP10 phage exhibited specificity for 66.7% of the ST307 strains. Furthermore, combining the three phages increased the host range by up to 85.7% (n = 36/42) against both the GC258 (80.7%, n = 21) and the ST307 (93.7%, n = 15) strains. These bacteriophages generally infected efficiently 94–100% of strains (EOP > 0.5) and controlled bacterial growth by up to 73.5% within 12 h. All bacteriophages belonged to the Caudoviricetes class and possessed characteristic Myovirus tails. They were also stable at temperatures between 4 and 50 °C and pH between 4 and 10 [8].
Regarding coliphages, the host range shows that FEC1 and FEC2 phages had a high specificity against strains of phylogroup B2 (35%), whereas FEC4 phage were only active against their host bacteria. The combination of three phages increased activity against strains of this phylogroup up to 65%. The efficiency of plating results showed efficient infection in 66.6–100% of strains (EOP > 0.5), and the infection curves enabled control of up to 94.5% of bacterial growth within the first three hours. The phages belonged to the Caudoviricetes class; two had a Sifovirus-type tail, while one presented Podo-virus morphology. All bacteriophages were stable at temperatures between 4 and 50 °C and pH levels between 4 and 12.

3.2. Bacterial Viability in Synthetic Wastewater

These experiments in wastewater demonstrated the maintenance of bacterial viability for 72 h. The CR-Kp strains KP58 (CG258-ST512), KH45 (ST307), and KR06 (CG258-ST258) presented initial concentrations of 5.18 ± 0.005 Log; 5.12 ± 0.018 Log and 5.06 ± 0.026 Log, respectively. These concentrations increased by approximately 2 logs at 18 h (7.03 ± 0.028 Log, KP58; 7.06 ± 0.025 Log, KH45 and 7.34 ± 0.017 Log, KR06) and remained viable until 72 h. (Figure 1a).
On the other hand, the ESBL-Ec strains HD67 (B2-phylogroup-blaCTX-M-G9), HD184 (E- phylogroup-blaCTX-M-G9), and HD163 (B2-phylogroup-blaCTX-M-G1) were added to the wastewater until initial concentrations of 5.25 ± 0.029 Log; 5.29 ± 0.013 Log and 5.24 ± 0.006 Log, respectively. These bacteria achieved concentrations of 7.39 ± 0.011 Log, 7.43 ± 0.012 Log and 7.38 ± 0.016 Log at 18 h post-administration, which remained stable until 72 h into the experiment (Figure 1b). These results made it possible to establish hour 18 (t18) as the optimum time for dosing the bacteriophages in the biocontrol assays because at this time, the bacteria showed stability in their concentrations.

3.3. Bacteriophage Stability in Synthetic Wastewater

The stability experiments demonstrated that the three phages remained stable for three hours in synthetic wastewater. The CR-Kp phages FKP3, FKP10, and FKP14 had initial concentrations of 8.44 ± 0.009 Log, 8.24 ± 0.008 Log, and 8.40 ± 0.033 Log, respectively. Both FKP10 and FKP14 phages decreased in concentration by 0.33 and 0.45 Log, respectively, during the first 3 h after dosing; however, these concentrations remained stable until 72 h (FKP10; 7.73 ± 0.029 Log to 7.94 ± 0.032 Log and FKP14; 7.93 ± 0.016 to 8.02 ± 0.022 Log). On the other hand, FKP3 reduced approximately 1 Log in the first 3 h, remaining stable until 72 h with concentrations between 7.25 ± 0.041 and 7.42 ± 0.016 Log (Figure 2a).
For bacteriophages targeting ESBL-Ec, the initial concentrations in wastewater were 8.43 ± 0.012 Log for FEC1: 8.28 ± 0.016 Log for FEC2 and 8.25 ± 0.079 Log for FEC4. Both FEC1 and FEC2 decreased by approximately 1 Log in the first 3 h after phage dosing and remained stable until 72 h (FEC1; 7.36 ± 0.012 to 7.42 ± 0.021 Log and FEC2; 7.33 ± 0.033 to 7.35 ± 0.015 Log). On the other hand, the FEC4 phage exhibited a reduction of approximately 2 Log during the first 3 h and stabilized later with concentrations between 6.18 ± 0.015 and 6.35 ± 0.055 Log (Figure 2b). Based on these bacterial viability results and previously published bacteriophage characterization assays, an MOI ratio of 10 (1 × 107 CFU/mL/1 × 108 CFU/mL) was established for all experiments.

3.4. Biocontrol Assay Using Individual Bacteriophages

Biocontrol experiments using individual bacteriophages demonstrated high killing efficiency against CR-Kp and ESBL-Ec isolates in wastewater. Phages FKP3 and FKP14 killed CR-Kp isolates belonging to CG258-ST512 (KP58) (75%) and CG258-ST258 (KR06) (87%), after 6 h of treatment. Furthermore, both bacteriophages eliminated 99.9% of the bacterial population at 54 h, with reductions of 2.99 Log (KP58) (p < 0.001) and 2.89 Log (KR06) (p < 0.001) (Figure 3a,b). Furthermore, the FKP10 bacteriophage eliminated the CR-Kp-ST307 strain with 99% (−2.39 Log) effectiveness after 36 h of treatment (p < 0.001) (Figure 3c). The bacteriophages after 54 h of treatment increase their concentrations in 2.5 Log (FKP3), 1.83 Log (FKP14), and 2.03 Log (FKP10) (Figure 3a–c).
On the other hand, biocontrol assays using E. coli phages produced killing efficiencies ranging from 87.9% to 99.2%. The FEC1 phage eliminated 90.2% of theESBL-Ec-blaCTX-M-G9 (HD67) population after 6 h of treatment, reaching a maximum bacterial reduction of 2.12 Log (99.2%) after 54 h (p < 0.001) (Figure 3d). Phages FEC2 and FEC4 effectively killed ESBL-Ec-blaCTX-M-G9 (HD184) and ESBL-Ec-blaCTX-M-G1 (HD163) strains with clearance percentages of 87.7% (−0.91 Log) and 90.0% (−1.0 Log), respectively, after 30 h (Figure 3e,f). In addition, bacteriophage FEC2 achieved a maximum reduction of 94.9% (−1.29 Log) after 36 h (p < 0.001). The quantification of bacteriophages after 54 h of treatment showed increases in their concentrations of 2.11 Log, 1.12 Log, and 1.12 Log for phages FEC1, FEC2, and FEC4, respectively (Figure 3d–f).

3.5. Biocontrol Assay Using Bacteriophage Cocktails

Biocontrol experiments using phage cocktails showed superior performance against some CR-Kp and ESBL-Ec strains compared to assays conducted with individual bacteriophages. In particular, the phage cocktail against CR-Kp (FKP3, FKP14, and FKP10) showed high effectiveness against the CG258 strains, resulting in a reduction of 85.2% (-0.83 Log) and 88.4% (−0.94 Log) in the first 6 h of treatment for the ST512 (KP58) and ST258 (KR06) strains, respectively. Furthermore, the cocktail eliminated 99.99% (−4.12 Log) (p < 0.001) of CR-Kp-ST512 (KP58) at 36 h and 99.9% (−3.20 Log) of CR-Kp- ST258 (KR06) after 54 h of treatment (p < 0.001) (Figure 3a,b). When the results of the cocktail versus the individual bacteriophage were compared, better performance of the cocktail was observed with the CR-Kp-CG258-ST512 (KP58) strain, which allowed an elimination up to 38.92% (1.16 Log) more than that obtained with the individual phage FKP3 (p < 0.001). In contrast, with the CR-Kp-CG258-ST258 (KR06) strain, the cocktail did not show differences in the elimination of the bacterial population versus individual bacteriophage action (FKP14) (Figure 3a,b). Moreover, for the CR-Kp-ST307 (KH45) isolate, the phage cocktail reduced 99% (−2.32) of the bacterial population after 54 h of treatment (p < 0.001), and at 30 h, it performed better than individual phages (FKP10), allowing the elimination of 65.31% more of the population (p < 0.001) (Figure 3c).
The use of the phage cocktail against ESBL-Ec (FEC1, FEC2 and FEC4) also showed better activity than the use of individual bacteriophages. This cocktail eliminated 93.1% (−1.16 Log) and 59.4% (−0.67 Log) strains carrying blaCTX-M-G9 (HD67 and HD184, respectively) at 6 h of treatment. Furthermore, both strains were efficiently eliminated by 99.6% (−2.52 Log; HD67) (p < 0.001) and 99.9% (−3.43 Log; HD184) (p < 0.001) after 54 and 36, respectively (Figure 3d,e). The cocktail also exhibited activity against ESBL-Ec-blaCTX-M-G1 (HD163), eliminating 92.94% (−1.15 Log) of the bacterial population after 54 h of treatment (p < 0.001) (Figure 3f). In particular, the cocktail against the HD67 strain (B-phylogroup-blaCTX-M-G9) reduced the population twice at 30 h (0.93 Log vs. 1.91 Log) compared with the use of the individual phage (p < 0.001); moreover, at 54 h of treatment, it still eliminated 18.89% (0.40 Log) more of the population (p < 0.001) (Figure 3d). Likewise, the cocktail showed a better performance with the host strain of the FEC2 phage (HD184; ST131 E-phylogroup-blaCTX-M-G9), and its activity at 6 h was 14 times higher (14,000%) (0.62 Log) than that observed with the individual phage (p < 0.001). Furthermore, it maintained a higher activity during the whole treatment, showing an increase in activity of 118%, 166%, and 231% more, compared with that observed with the individual phage at times 30 (p < 0.001), 36 (p < 0.001), and 54 (p < 0.001), respectively (Figure 3e). In contrast, the cocktail showed no differential activity compared with the individual phage when evaluated against the host strain of phage FEC4 (Figure 3f).

3.6. Biocontrol Assays Using Multiple Doses of the Cocktail

Bacterial biocontrol experiments using multiple doses of the phage cocktail showed a slight increase in log reduction compared with the results obtained using a single dose of the cocktail. In biocontrol assays with the CR-Kp strain of CG258-ST512 (KP58), a 1.10 log reduction (92.03%) was observed after one dose. After two doses of the cocktail, a reduction of 4.08 logarithms (99.99%) was observed at 30 h, indicating an increase in clearance of 15.5% (0.63 Log) compared with the administration of the cocktail in a single dose at the same time (p = 0.53) Finally, after administering the three doses of the cocktail, 99.99% (4.26 Log) of the bacterial population was eliminated at 36 h (p < 0.001); however, at 54 h of treatment, the population increased by 1.19 Log. In this sense, the logarithmic reduction after 54 h of treatment with several doses of the cocktail was lower (−3.07 Log) than that observed with the single-dose cocktail treatment (−3.90 Log) (Figure 4a).
In contrast, with the CR-Kp-ST307 (KH45) strain, a better performance of the cocktail was evidenced with multiple doses during the whole treatment time, which meant an elimination of 97.25% (−1.56 Log) after one dose; 99.96% (−3.48 Log) with two doses (p < 0.001) and 99.96% (−3.50 Log) with three doses (p < 0.001). These results show an increase in clearance between 155% and 186% more than that observed with single-dose cocktail administration, showing a maximum efficacy after 54 h of treatment (99.97%; −3.59 Log) (p < 0.001). (Figure 4b).
Biocontrol assays using ESBL-Ec-ST131-blaCTXM-G1 (HD163) showed elimination of 98.2% (−1.76 Log) of the bacterial population at 30 h. After the addition of two doses of the cocktail, bacterial clearance increased by 68.2% (0.71 Log) at 30 h compared with the performance of the single-dose cocktail at the same time (p < 0.001). However, after the third dosing, the bacterial population rapidly regrew to 7.17 Log at 54 h of treatment (Figure 4c).

3.7. Biocontrol Assays Using Individual and Sequential Administration of the Bacteriophages Composing the Cocktail

Biocontrol experiments using individual and sequential administration of the cocktail phages against the CR-Kp-CG258-ST512 (KP58) strain showed similar results to those obtained with the use of the cocktail in three doses. Administration of the FKP3 phage at time zero allowed a reduction of 1.26 (94.5%) logarithms at 6 h, and the addition of phage FKP14 resulted in a reduction of 4.3 (99.99%) logarithms at 36 h of treatment. In this sense, the use of the phages of the cocktail administered individually and sequentially increased the elimination of the population by 16.6% (0.573) at 30 h, compared to the administration of the cocktail in a single dose (p < 0.001). However, with the use of individual and sequential phages, a regrowth of the bacterial population of 1.17 logarithms was observed after 54 h of treatment; therefore, during this time, the performance of the single-dose cocktail was better (−3.9 Log) than the performance of the sequentially administered phage (−3.1 Log) (Figure 5a).
The individual and sequential administration of the phages of the cocktail against ESBL-Ec-bla-CTX-M-G1 (HD163) produced results like those observed with the administration of the cocktail in a single dose. After dosing the FEC1 bacteriophage, a 6.8% (−0.067 Log) reduction was observed after 6 h, and the effectiveness increased to 89.44% (−1.04 log) after 30 h of treatment with the addition of the FEC2 phage (p < 0.001). Finally, the addition of FEC4 allowed a 92.21% (−1.10 Log) elimination at 36 h; however, a regrowth of 1.09 logarithms of the bacterial population was observed in the wastewater after 54 h of treatment (Figure 5b). In this strain, the use of single phages administered sequentially was less efficient (89.44%, −1.044 l Log) at 30 h of treatment compared with the use of the cocktail administered in three doses (98.2%, −1.76 Log) (p < 0.001). However, after 54 h, the administration of the cocktail in a single dose (91.41%, −1.06 Log) (p < 0.001) was more effective than the use of the cocktail in multiple doses (3.49%, −0.015 Log) (p = 0.202) and then the administration of the cocktail phages sequentially (3.81%, −0.017 Log) (p = 0.181) (Figure 5b).

3.8. Assessment of Bacterial Resistance to Bacteriophages

During wastewater treatment, we identified morphological changes in Klebsiella pneumoniae and E. coli colonies, including reduced colony size and irregular borders (Figure 6). These changes occurred earlier with individual bacteriophage treatments than with the cocktail. For the CR-Kp strains (KP58 and KR06), smaller colonies appeared after 30 h with individual phages and after 36 h with a single-dose cocktail, multi-dose cocktail, or sequential cocktail treatments. In CR-Kp-ST307 (KH45), irregular borders emerged after 30–36 h and earlier when treated with multiple cocktail doses. In ESBL-Ec strains (HD67, HD184), similar changes appeared after 30 h with single phages and after 36 h with the cocktail, whereas in HD163, changes occurred as early as 6 h. Despite these morphological changes, spot tests confirmed that the selected strains developed resistance to both individual phages and the cocktail, including some KP58 subpopulations that remained morphologically unchanged but were also resistant. Finally, three phage-resistant CR-Kp strains (KP58, KR06, and KH45) were cultured on CROMID®CARBA media to confirm carbapenem resistance. All strains grew and exhibited morphological changes (small size or irregular borders) with variations in chromogenic compound hydrolysis. Antibiotic susceptibility testing of KP58 and KH45 showed minor MIC changes, but resistance profiles remained largely unchanged. KP58 remained resistant to multiple antibiotic classes, including β-lactams, quinolones, aminoglycosides, nitrofurantoin, and TMP-SMX, whereas KH45 retained resistance to β-lactams, quinolones, and TMP-SMX.

4. Discussion

Global and local studies have consistently identified wastewater as a critical hotspot for the dissemination of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Acting as both a dynamic vector and reservoir, wastewater accumulates a wide array of biological and chemical contaminants originating from urban, hospital, industrial, and agricultural sources. As a result, wastewater treatment plants (WWTPs) receive and concentrate high loads of ARB, antibiotics, and their metabolites, creating ideal conditions for microbial interactions, selective pressure, and horizontal gene transfer—particularly in the presence of sub-inhibitory concentrations of antimicrobials. Although conventional and advanced treatment processes in WWTPs effectively reduce total microbial load and organic matter, they are often insufficient to eliminate ARB and ARGs entirely. This limitation allows their persistence, potential amplification, and subsequent release into surface waters. Therefore, improving wastewater treatment strategies is essential to mitigate the environmental and public health risks associated with antimicrobial resistance. [2,4,21]. This study demonstrated the potential of bacteriophages as biocontrol tools for the selective elimination of antibiotic-resistant pathogenic bacteria in synthetic wastewater. In addition, it contributes to the development of further research for the improvement of traditional tertiary wastewater treatment systems, enabling the safe reuse or discharge of treated water to the environment.
Although bacteriophages have been studied for over a century, their application as biocontrol agents in wastewater systems has only gained attention in the last 15 years. Since 2011, research has focused on using phages to address operational problems such as bulking, foaming, and odor production by targeting bacterial genera like Corynebacterium, Gordonia, Nocardia, Haliscomenobacter, and Pseudomonas putida [22,23,24]. More recently, bacteriophages have also been evaluated in combination with physical methods, such as UV treatment, to control biofilm-forming bacteria in anaerobic bioreactor membranes [22]. However, experiments based on the use of bacteriophages for the biocontrol of antibiotic-resistant pathogens in wastewater are even scarcer. This study pioneers phage-based biocontrol against β-lactam-resistant bacteria in wastewater, showing high efficacy up to 99.99% reduction against clinically and epidemiological relevant K. pneumoniae (CG258-ST512/ST258, ST307) and E. coli (ST131 clone, phylogroups B2/E), including KPC and ESBL producers, respectively. These findings agree with some publications that have focused on eliminating antimicrobial-resistant pathogens in wastewater [23,24,25]. According to a study by Sadeqi S. et al. [26], the use of a bacteriophage cocktail allowed the elimination of 99.8% of the population of K. pneumoniae and E. coli producing ESBL in primary sedimentation tanks of a hospital wastewater treatment system. Likewise, another study published in 2023 demonstrated the effectiveness of a bacteriophage cocktail for the elimination of 99.6% of the multidrug-resistant E. coli population after 24 h of treatment of a raw wastewater effluent from Egypt [24]. In addition, studies conducted against ESBL-producing K. pneumoniae have found high effectiveness of jumbo bacteriophages in controlling approximately 90% (−1.25 Log) of the bacterial population in wastewater during 8 h of treatment [27].
These publications show a greater interest in the biocontrol of microorganisms producing extended-spectrum β-lactamases (ESBL) or described as multiresistant; however, research on biocontrol strategies targeting carbapenem-resistant pathogens remains limited [23]. One study published by the University of Houston, Texas, reported a 2.4 log reduction of a population of NDM carbapenemase-producing E. coli using polyvalent phages in activated sludge (WWTP aeration tanks) [28], while no biocontrol studies against carbapenemase-producing K. pneumoniae have been reported to date. In contrast, other researchers have focused on exploring biocontrol strategies against other pathogens, such as Salmonella typhi, S. enterica, antibiotic-susceptible E. coli, Pseudomonas aeruginosa among others, mainly in domestic wastewater, hospital wastewater, and sewage sludge matrices (WWTP) [23,25,29]. In addition, biocontrol studies have been conducted on drinking water, rainwater, and irrigation water [15,16,17]. In this sense, the results of this study are of great relevance, considering that bacteriophage-based treatments achieved effective elimination against antibiotic-resistant bacteria that have a major impact on health care-associated infections and have been recognized as a critical threat by the WHO (CR-Kp-blaKPC and ESBL-Ec) [3,7]. Moreover, these bacteria are widely disseminated in environmental settings worldwide and are highly persistent [2,19].
On the other hand, in this study, a single dose of cocktail performed equally or better than the use of individual bacteriophages, which enhanced activity by up to 65.31% and 14-fold against (14,000%) CR-Kp and ESBL-Ec, respectively. These findings coincide with studies carried out in wastewater with different bacterial models, which have reported bacterial elimination efficacies of the cocktails between 90% and 99.9% [16,17,23,24,27]; however, unlike this work, other authors did not compare the administration of the cocktail in different doses and few publications compare the performance of individual phages versus the cocktail in biocontrol assays in wastewater [30,31]. In addition, studies evaluating the administration of phages individually and sequentially have been conducted in in vitro and in vivo models [32]. Although phage cocktails are generally considered more effective than individual phages due to broader host range and reduced resistance, their success depends on multiple factors, including the type of bacteriophages using the selection criteria for the inclusion of the phages in the cocktail, the multiplicity of infection, and the time and form of administration, among others [33,34]. Our findings showed that the phage cocktail was more effective at lysing clinical isolates of K. pneumoniae and E. coli than individual phages. This effect may be due to strain-specific differences in susceptibility to phages, resulting from clonal variation in surface receptors, which enables the cocktail to cover a broader host range. Additionally, phages in the cocktail could exhibit additive or synergistic interactions during co-infection, enhancing bacterial lysis beyond that achieved by individual phages. Finally, the use of cocktails reduces the likelihood of resistance development, as simultaneous escape from multiple phages is less probable. Although receptor-binding protein characterization was not performed, future work will include RBP and receptor mapping to better elucidate these mechanisms [29].
Likewise, this study proposes several cocktail dosage alternatives to improve bacterial elimination and decrease the appearance of phage-resistant populations. The present results showed that the use of the cocktail in a single dose allowed a delay in the regrowth of phage-resistant populations during the 54 h of treatment, compared with the administration of multiple doses of the cocktail and the phages of the cocktail administered individually and sequentially; experiments where an increase in phage-resistant subpopulations was observed from 6 (ESBL-Ec), 30, and 36 (CR-Kp) hours. These findings coincide with other publications in which the emergence of phage-resistant subpopulations has been reported with the use of cocktails. However, the times of resistance emergence were variable in the studies [15,16,17,23,24,27]. On the other hand, experiments of applying multiple doses of cocktail and administering sequential bacteriophages individually for wastewater biocontrol are not common; however, in other types of applications, there has been much questioning about the most effective form of cocktail administration [32]. According to Hall R et al. [29], simultaneous administration of multiple phages may be more effective than sequential application if there is a low probability that bacteria will become resistant in a single step (if it requires several different mutations) or if multidrug resistance to phages is costly to the bacteria. On the other hand, if bacteria can acquire resistance to any number of phages relatively easily, sequential application may be more effective, continuously exposing bacteria to phages to which they are not initially resistant and keeping the bacterial population density low for longer [29]. These hypotheses could correlate with our results, considering that the one-dose cocktail was the most effective and the emergence of resistant bacteria occurred later. In addition, the populations of phage-resistant bacteria showed alterations in colony morphology and size, which could indicate the biological cost of bacteriophage resistance (without antibiotic re-sensitization), as described in other studies [33]. Therefore, it is of great importance to extrapolate concepts of clinical phage therapy into the field of environmental biocontrol, using different forms of phage and cocktail administration to observe their performance under different conditions and optimize their use in different applications. In addition, it is necessary to determine evolutionary patterns in complex matrices, such as wastewater.
Regarding the experimental conditions in the biocontrol trials, this study evaluated the treatments for 54 h; an aspect that is relevant not only to identify the time of effectiveness but also to determine the appearance of subpopulations resistant to the phages. Biocontrol trials in wastewater published in the literature have reported different treatment times that vary from 6 months to 120, 96, 24, and 8 h [15,16,17,23,24]. However, it is not possible to define an ideal time for all trials, and it should be in accordance with the application times in real matrices according to each proposed treatment system. In particular, the use of bacteriophages has been proposed as part of the treatment systems of wastewater treatment plants (WWTPs), and the success of their application in these locations depends on different factors. In this sense, it is important to identify the pathogens to be eliminated with treatment and to confirm the susceptibility of these bacteria to the phages. In addition, effective concentrations and doses of application should be determined considering the average bacterial density in the water [31]. Other relevant aspects include the operational conditions of the plant, such as flow, retention times, and the use of flocculants and disinfectants, among others, in addition to identifying whether the bacteriophages have the capacity to penetrate flocs and maintain effectiveness under in situ conditions [33,34]. Considering the above, there is a need to perform full-scale research to determine the best operational conditions to obtain plausible results at the time of implementation for treating real wastewater; in this sense, a potential approach includes applying bacteriophages in the final effluent to minimize the release of antibiotic-resistant bacteria into the environment.
On the other hand, although this study uses synthetic wastewater, these results show a high effectiveness of bacteriophages for the elimination of CR-Kp and ESBL-Ec in simulated wastewater matrices with pH and organic matter loads, similar to those found in domestic wastewater [35]. Several publications have used simulated microenvironments like ours to perform biocontrol tests, which are based on the use of real matrices that undergo filtration and sterilization processes to eliminate bacterial populations that could interfere in the evaluations [23,31]. Conversely, studies performed in real matrices are more limited and require the combination of phenotypic and molecular methodologies for the evaluation of the effectiveness of the treatments. In general, studies carried out on real wastewater pose substantial challenges for initial evaluations due to its high variability in composition, which depends on factors such as its origin, local treatment conditions, and seasonal fluctuations. These factors influence parameters like organic and inorganic loads, suspended solids, and microbial diversity. It is for this reason that it is crucial to assess the stability, viability, and infectivity of bacteriophages directly in real wastewater matrices. Such evaluations are necessary to understand phage performance in the presence of diverse microbial communities and under fluctuating physicochemical conditions, including variations in temperature, pH, organic content, and the presence of potential inhibitors. Furthermore, it is essential to assess the impact of phage application on native microbial communities to ensure high host specificity and avoid unintended ecological disturbances within wastewater treatment plants (WWTPs) [24,27]. This work is the starting point for the execution of projects that determine the effectiveness of bacteriophages in real environmental matrices; to determine if interference exists of the bacterial communities in the treatment; and to confirm that the natural bacterial populations are not affected by the action of bacteriophages.
Conventional wastewater disinfection methods, such as chlorination, UV irradiation, and ozonation, are widely used due to their relatively low cost (USD 0.02–0.04/m3); however, they exhibit important limitations when addressing antimicrobial resistance. High chlorine doses can effectively reduce bacterial loads but may also select chlorine-resistant strains [36], while UV treatment, although efficient, is energy-intensive and may permit bacterial DNA repair and reactivation [37]. Moreover, these methods lack specificity for antibiotic-resistant bacteria (ARB) and do not mitigate the dissemination of antibiotic resistance genes (ARGs). In response, alternative eco-friendly strategies—such as constructed wetlands, membrane bioreactors, biochar-based adsorption, and photocatalysis—have emerged, offering broader benefits including the removal of ARB, degradation of residual antibiotics, and reduction of ARGs. In this context, bacteriophage-based biocontrol represents a promising and sustainable strategy due to its high specificity, capacity to replicate in the presence of host bacteria, and lack of harmful or mutagenic by-products. Phages can selectively lyse antimicrobial-resistant bacterial strains, reducing the bacterial populations most associated with horizontal gene transfer, and thus contribute to limiting ARG propagation in treated effluents. Their formulation stability can be improved through encapsulation or lyophilization, and their integration into existing wastewater infrastructure is technically feasible. Although a full techno-economic evaluation was beyond the scope of this study, current data suggest that phage-based approaches could complement or enhance traditional disinfection systems, offering a cost-effective solution with lower ecological impact and the potential to reduce hidden public health costs.
Finally, considering the limitations of wastewater treatment systems for eliminating resistant bacteria [38], the results of this study provide strong experimental evidence supporting the use of bacteriophage cocktails as an effective and environmentally friendly strategy to control the spread of antimicrobial resistance in aquatic environments, in order to improve water quality and allow its reuse in different human and animal activities. Furthermore, these treatment approaches could be optimized by searching for other bacteriophages with different receptor-binding proteins that eliminate resistant subpopulations or by using them in combination with other disinfection methodologies (tertiary treatments), such as chlorination, UV light, and the use of advanced oxidation processes, among others [36]. Treatments based on bacteriophages, in addition to reducing the presence of resistant bacteria, also reduce the use of chemical products in water, which has a positive impact on the environment.
Considering the importance of these results and the prospects for further research, we propose key considerations for advancing phage-based biocontrol in wastewater treatment plants (WWTPs). Developing research from these perspectives would help overcome the limitations of using bacteriophages in wastewater treatment. First, it is essential to assess phage stability, viability, and infectivity directly in real wastewater matrices, given their complex microbial diversity and variable physicochemical conditions. In addition, the persistence of phage should be monitored, and decay rates must also be measured to optimize dosing strategies. In parallel, the impact of phage application on native microbial communities must be monitored to ensure host specificity and prevent ecological disruptions.
Future work should progress from laboratory studies to pilot-scale trials in WWTPs, especially in critical streams like hospital effluents, integrating qPCR and metagenomics to monitor ARB, ARGs, and microbial community shifts. Additionally, it is important to track horizontal gene transfer (HGT) to avoid unintended ARG dissemination. Finally, the development of regulatory frameworks and biosafety protocols, alongside continuous monitoring of ARB, ARGs, and phages, is necessary to ensure the safe and effective environmental application of phage-based treatments.

5. Conclusions

These results demonstrate the high efficacy of bacteriophages (99%) and phage cocktails (99.99%) in reducing the bacterial populations of CR-Kp (KPC) and ESBL-Ec in simulated laboratory-scale wastewater. In addition, the cocktail optimization assays showed that the administration of the cocktail in a single dose was the most effective treatment because of the high reduction percentage and the prevention of the early appearance of phage-resistant subpopulations. This is a pioneering study of the use of bacteriophages as biocontrol agents for ARBs in simulated laboratory-scale wastewater in the country, demonstrating their potential for the control of clinically important bacteria of local and global interest. The results open a window of opportunity to consolidate the use of phages as an innovative, sustainable, and complementary strategy to improve water quality, allowing its safe reuse.
This eco-friendly and highly specific approach offers an alternative to conventional disinfection methods and holds potential for integration into risk-based water quality management, particularly in scenarios involving water reuse or the agricultural application of biosolids. Thus, this study supports proactive environmental interventions and highlights the need to incorporate AMR mitigation into public health policies and national action plans. From a public health perspective, our findings offer a scientific basis for further integrating bacteriophage-based treatments into advanced wastewater management systems, with the potential to reduce selective pressures and limit the environmental dissemination of AMR. Incorporating phage-based strategies into AMR control aligns with the One Health framework by simultaneously safeguarding human, animal, and environmental health. This approach can help bridge the gap between environmental management and clinical interventions, promoting a more integrated and sustainable solution to AMR.

Author Contributions

Conceptualization, M.D.Z.-M., L.S.-O. and J.N.J.; formal analysis, M.D.Z.-M. and L.S.-O.; funding acquisition, J.N.J.; investigation, M.D.Z.-M., L.S.-O. and J.N.J.; methodology, M.D.Z.-M., L.S.-O. and J.N.J.; project administration, J.N.J.; supervision, L.S.-O. and J.N.J.; validation, M.D.Z.-M. and L.S.-O.; visualization, M.D.Z.-M. and L.S.-O.; writing—original draft, M.D.Z.-M., L.S.-O. and J.N.J.; writing—review and editing, M.D.Z.-M., L.S.-O. and J.N.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministerio de Ciencia y Tecnología de Colombia [MINCIENCIAS], grant number 111589785393, 2021.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

ARBAntimicrobial-resistant bacteria
ARGsAntibiotic resistance genes
CR-KpCarbapenem-resistant Klebsiella pneumoniae
ESBL-EcE. coli producers of extended spectrum β-lactamases
KPCKlebsiella pneumoniae carbapenemase
MLSTMulti-Locus Sequence Typing
WHOWorld Health Organization
WWTPsWastewater treatment plants

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Figure 1. Bacterial viability in synthetic sewage. The figure shows the bacterial viability of phage host strains in wastewater for 72 h. (a) shows the bacterial viability of the strains of CR-Kp belonging to CG258-ST512 (KP58), CG258-ST258 (KR06), and ST307 (KH45). The three strains are the host bacteria of the phages FKP3 (KP58), FKP14 (KR06), and FKP10 (KH45). (b) shows the bacterial viability of the ESBL-Ec strains from B2-phylogroup-blaCTX-M-G9 (HD67), E- phytogroup-blaCTX-M-G9 (HD184), and B2-phylogroup-blaCTX-M-G1 (HD163), which are the host strains of phages FEC1, FEC2, and FEC4, respectively.
Figure 1. Bacterial viability in synthetic sewage. The figure shows the bacterial viability of phage host strains in wastewater for 72 h. (a) shows the bacterial viability of the strains of CR-Kp belonging to CG258-ST512 (KP58), CG258-ST258 (KR06), and ST307 (KH45). The three strains are the host bacteria of the phages FKP3 (KP58), FKP14 (KR06), and FKP10 (KH45). (b) shows the bacterial viability of the ESBL-Ec strains from B2-phylogroup-blaCTX-M-G9 (HD67), E- phytogroup-blaCTX-M-G9 (HD184), and B2-phylogroup-blaCTX-M-G1 (HD163), which are the host strains of phages FEC1, FEC2, and FEC4, respectively.
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Figure 2. Bacteriophage stability in synthetic sewage. The figure shows the results of the phage stability assays in the simulated wastewater after 72 h. (a) presents the stability of the CR-Kp phages (FKP3, FKP10, and FKP14) in synthetic sewage, and (b) presents the stability of the ESBL-Ec phages (FEC1, FEC2, and FEC4).
Figure 2. Bacteriophage stability in synthetic sewage. The figure shows the results of the phage stability assays in the simulated wastewater after 72 h. (a) presents the stability of the CR-Kp phages (FKP3, FKP10, and FKP14) in synthetic sewage, and (b) presents the stability of the ESBL-Ec phages (FEC1, FEC2, and FEC4).
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Figure 3. Elimination curves of CR-Kp and ESBL-Ec in wastewater using individual bacteriophages and a phage cocktail. Figure shows the bacterial elimination curves in wastewater after 54 h of treatment using individual bacteriophages and phage cocktails. (a–c) show the biocontrol assays on CR-Kp strains belonging to CG258-ST512 (KP58) (a); CG258-ST258 (KR06) (b) and ST307 (KH45) (c); and (d–f) show the biocontrol assays on ESBL-Ec strains belonging to B2-phylogroup-blaCTX-M-G9 (HD67) (d), E-phytogroup-blaCTX-M-G9 (HD184) (e), and B2-phylogroup-blaCTX-M-G1 (HD163) (f). The figures in the small boxes show the logarithmic reduction for each treatment. Each experiment included a bacteriophage stability assay. The FKP3 and FKP14 phages maintained stable concentrations until time 54, with values of 6.40 × 107 PFU/mL and 1.83 × 108 PFU/mL, respectively. The FKP10 phage maintained a stable concentration of 2.57 × 108 PFU/m until time 54. The coliphages FEC1 and FEC2 maintained stable concentrations until time 54, with values of 1.55 × 108 PFU/mL and 1.58 × 108 PFU/mL, respectively. In FEC4, the bacterial concentration remained stable from 30 to 54 h.
Figure 3. Elimination curves of CR-Kp and ESBL-Ec in wastewater using individual bacteriophages and a phage cocktail. Figure shows the bacterial elimination curves in wastewater after 54 h of treatment using individual bacteriophages and phage cocktails. (a–c) show the biocontrol assays on CR-Kp strains belonging to CG258-ST512 (KP58) (a); CG258-ST258 (KR06) (b) and ST307 (KH45) (c); and (d–f) show the biocontrol assays on ESBL-Ec strains belonging to B2-phylogroup-blaCTX-M-G9 (HD67) (d), E-phytogroup-blaCTX-M-G9 (HD184) (e), and B2-phylogroup-blaCTX-M-G1 (HD163) (f). The figures in the small boxes show the logarithmic reduction for each treatment. Each experiment included a bacteriophage stability assay. The FKP3 and FKP14 phages maintained stable concentrations until time 54, with values of 6.40 × 107 PFU/mL and 1.83 × 108 PFU/mL, respectively. The FKP10 phage maintained a stable concentration of 2.57 × 108 PFU/m until time 54. The coliphages FEC1 and FEC2 maintained stable concentrations until time 54, with values of 1.55 × 108 PFU/mL and 1.58 × 108 PFU/mL, respectively. In FEC4, the bacterial concentration remained stable from 30 to 54 h.
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Figure 4. Elimination curves of CR-Kp and ESBL-Ec in wastewater after the administration of multiple doses of the phage cocktail. Figure 4 shows the bacterial elimination curves in wastewater during 54 h of treatment using phage cocktails added at the beginning of the experiment (t0) and at 6 and 30 h after the beginning of the experiment. Figure 4a,b show the biocontrol assays on CR-Kp strains belonging to CG258-ST512 (KP58) (a) and ST307 (KH45) (b), and (c) shows the biocontrol assays on ESBL-Ec strains belonging to the B2-phylogroup blaCTX-M-G1 (HD163). The black arrows indicate the administration of each dose of the cocktail. The quantification data in the figure corresponds to the number of bacteria obtained before the administration of each dose of the cocktail. The figures in the small boxes show the logarithmic reduction for each treatment.
Figure 4. Elimination curves of CR-Kp and ESBL-Ec in wastewater after the administration of multiple doses of the phage cocktail. Figure 4 shows the bacterial elimination curves in wastewater during 54 h of treatment using phage cocktails added at the beginning of the experiment (t0) and at 6 and 30 h after the beginning of the experiment. Figure 4a,b show the biocontrol assays on CR-Kp strains belonging to CG258-ST512 (KP58) (a) and ST307 (KH45) (b), and (c) shows the biocontrol assays on ESBL-Ec strains belonging to the B2-phylogroup blaCTX-M-G1 (HD163). The black arrows indicate the administration of each dose of the cocktail. The quantification data in the figure corresponds to the number of bacteria obtained before the administration of each dose of the cocktail. The figures in the small boxes show the logarithmic reduction for each treatment.
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Figure 5. Elimination curves of CR-Kp and ESBL-Ec in wastewater following the sequential administration of phages from the cocktail. Figure 5 shows the bacterial elimination curves in wastewater during 54 h of treatment after individual and sequential administration of the cocktail bacteriophages. (a) shows the biocontrol assays of the CR-Kp-CG258-ST512 (KP58) strain. FKP3 phage was administered at time zero (t0) and then FKP14 phage was added at 30 h. (b) presents the biocontrol assay results for ESBL-Ec strains belonging to the B2-phylogroup blaCTX-M-G1 (HD163). Phage FEC1 was administered at time zero, followed by phage FEC2 at six hours and phage FEC4 at 30 h. The black arrows indicate the time of bacteriophage administration. The quantification data in the figure corresponds to the number of bacteria obtained before the administration of each phage dose. The figures in the small boxes show the logarithmic reduction for each treatment.
Figure 5. Elimination curves of CR-Kp and ESBL-Ec in wastewater following the sequential administration of phages from the cocktail. Figure 5 shows the bacterial elimination curves in wastewater during 54 h of treatment after individual and sequential administration of the cocktail bacteriophages. (a) shows the biocontrol assays of the CR-Kp-CG258-ST512 (KP58) strain. FKP3 phage was administered at time zero (t0) and then FKP14 phage was added at 30 h. (b) presents the biocontrol assay results for ESBL-Ec strains belonging to the B2-phylogroup blaCTX-M-G1 (HD163). Phage FEC1 was administered at time zero, followed by phage FEC2 at six hours and phage FEC4 at 30 h. The black arrows indicate the time of bacteriophage administration. The quantification data in the figure corresponds to the number of bacteria obtained before the administration of each phage dose. The figures in the small boxes show the logarithmic reduction for each treatment.
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Figure 6. Morphological changes in CR-Kp and ESBL-Ec colonies after biocontrol treatments with bacteriophages in wastewater. Figure 6 shows the morphological changes of the bacteria during treatment with bacteriophages in wastewater. (a) corresponds to the colonies of CR-Kp in the control treatment, colonies after treatment with decreased size (b), and colonies after treatment with irregular edges (c,d). (e) corresponds to ESBL-Ec colonies in the control treatment, colonies after treatment with decreased size (f), and colonies after treatment with irregular borders (g,h).
Figure 6. Morphological changes in CR-Kp and ESBL-Ec colonies after biocontrol treatments with bacteriophages in wastewater. Figure 6 shows the morphological changes of the bacteria during treatment with bacteriophages in wastewater. (a) corresponds to the colonies of CR-Kp in the control treatment, colonies after treatment with decreased size (b), and colonies after treatment with irregular edges (c,d). (e) corresponds to ESBL-Ec colonies in the control treatment, colonies after treatment with decreased size (f), and colonies after treatment with irregular borders (g,h).
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Table 1. Characteristics of the bacteria used in the biocontrol assays.
Table 1. Characteristics of the bacteria used in the biocontrol assays.
Bacterial GenusStrain CodeSource β-Lactamases (Carbapenemases or ESBL)MLST Typing/Clermont Typing *Antimicrobial Resistant Profile
Carbapenem-resistant
K. pneumoniae		KP58Hospitalized patients infectionsKPC3CG258-ST512Resistant to ampicillin/sulbactam, piperacillin/tazobactam, cefoxitin, ceftazidime, ceftriaxone, cefepime, ertapenem, imipenem, meropenem,
amikacin, ciprofloxacin, tigecycline, and colistin. It also has intermediate
resistance to gentamicin.
KR06KPC2CG258-ST258Resistant to ampicillin/sulbactam, piperacillin/tazobactam, cefoxitin, ceftazidime, ceftriaxone, cefepime, ertapenem, imipenem, meropenem,
doripenem, amikacin, gentamicin, and ciprofloxacin.
KH45KPC2ST307Resistant to piperacillin/tazobactam, ceftazidime, cefepime, ertapenem, imipenem, meropenem, and ciprofloxacin. It also has intermediate resistance to cefoxitin, and is susceptible to amikacin and gentamicin.
E. coli resistant to third-generation cephalosporins
(ESBL)		HD67Colonized patientsCTX-M-G9Non-typing B2-phylogroupPositive ESBL test, resistant to ceftazidime, ceftriaxone, cefepime, and
ciprofloxacin. Intermediate resistance to colistin, and susceptible to ampicillin/
sulbactam, piperacillin/tazobactam, cefoxitin, ertapenem, imipenem,
meropenem, doripenem, amikacin, gentamicin, and tigecycline.
HD184CTX-M-G9ST131
E-phylogroup
HD163CTX-M-G1ST131
B2-phylogroup		Positive ESBL test, resistant to ceftazidime, ceftriaxone, and cefepime. Intermediate resistance to colistin, and susceptible to ampicillin/sulbactam,
piperacillin/tazobactam, cefoxitin, ertapenem, imipenem, meropenem,
doripenem, amikacin, gentamicin, ciprofloxacin, and tigecycline.
ESBL: extended-spectrum β-lactamases. MLST: Multilocus sequence typing. * For E. coli strains.
Table 2. Chemical composition of synthetic wastewater with domestic wastewater characteristics.
Table 2. Chemical composition of synthetic wastewater with domestic wastewater characteristics.
ComponentFinal Concentration on the Simulated Wastewater
(mg/L)(M)
Sodium chloride (NaCl) 7119
Potassium chloride (KCl) 454
Calcium chloride dihydrate (CaCl2· 2H2O) 427
Sodium bicarbonate (NaHCO3) 961.1
Calcium sulfate dihydrate (CaCO4·2H2O) 60348
Magnesium sulfate heptahydrate (MgSO4· 7H2O) 125507
Dibasic potassium phosphate (K2HPO4) 28161
Urea (CH4N2O) 699.9
Meat Extract (Carbon source)32-
Peptone (Nitrogen source)22-
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Zapata-Montoya, M.D.; Salazar-Ospina, L.; Jiménez, J.N. Combating Environmental Antimicrobial Resistance Using Bacteriophage Cocktails Targeting β-Lactam-Resistant High-Risk Clones of Klebsiella pneumoniae and Escherichia coli in Wastewater: A Strategy for Treatment and Reuse. Water 2025, 17, 2236. https://doi.org/10.3390/w17152236

AMA Style

Zapata-Montoya MD, Salazar-Ospina L, Jiménez JN. Combating Environmental Antimicrobial Resistance Using Bacteriophage Cocktails Targeting β-Lactam-Resistant High-Risk Clones of Klebsiella pneumoniae and Escherichia coli in Wastewater: A Strategy for Treatment and Reuse. Water. 2025; 17(15):2236. https://doi.org/10.3390/w17152236

Chicago/Turabian Style

Zapata-Montoya, María D., Lorena Salazar-Ospina, and Judy Natalia Jiménez. 2025. "Combating Environmental Antimicrobial Resistance Using Bacteriophage Cocktails Targeting β-Lactam-Resistant High-Risk Clones of Klebsiella pneumoniae and Escherichia coli in Wastewater: A Strategy for Treatment and Reuse" Water 17, no. 15: 2236. https://doi.org/10.3390/w17152236

APA Style

Zapata-Montoya, M. D., Salazar-Ospina, L., & Jiménez, J. N. (2025). Combating Environmental Antimicrobial Resistance Using Bacteriophage Cocktails Targeting β-Lactam-Resistant High-Risk Clones of Klebsiella pneumoniae and Escherichia coli in Wastewater: A Strategy for Treatment and Reuse. Water, 17(15), 2236. https://doi.org/10.3390/w17152236

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