Investigating the Effect of Zinc Salts on Escherichia coli and Enterococcus faecalis Biofilm Formation

Abstract

Water supply and sewage drainage pipes have a critical role to play in the provision of clean water and sanitation, and pipe material selection influences infrastructure life, water quality, and microbial communities. Zinc-containing compounds are highly valued due to their mechanical properties, anticorrosion behavior, and antimicrobial properties. However, the effect of zinc salts, such as zinc sulfate heptahydrate and zinc chloride, on biofilm-forming bacteria, including Escherichia coli and Enterococcus faecalis, is not well established. This study investigates the antibacterial properties of these zinc salts under simulated pipeline conditions using minimum inhibitory concentration assays, biofilm production assays, and antibiotic sensitivity tests. Findings indicate that zinc chloride is more antimicrobial due to its higher solubility and bioavailability of Zn²⁺ ions. At higher concentrations, zinc salts inhibit the development of a biofilm, whereas sub-inhibitory concentrations enhance the growth of biofilm, suggesting a stress response in bacteria. zinc chloride also enhances antibiotic efficacy against E. coli but induces resistance in E. faecalis. These findings highlight the dual role of zinc salts in preventing biofilm formation and modulating antimicrobial resistance, necessitating further research to optimize material selection for water distribution networks and mitigate biofilm-associated risks in pipeline systems.

1. Introduction

Water supply and sewage drainage systems are crucial for providing safe and clean water and efficient sanitation. Pipe material selection is a fundamental component in determining not only the mechanical durability and lifespan of such systems but also their impact on water quality and microbial populations that develop in them. Of the various materials used in pipelines, metals such as iron, copper, and zinc are used as they are corrosion-resistant, mechanically strong, and possess natural antimicrobial properties. Zinc and its salts have been widely used in various applications, including galvanized steel paint, antifouling paint, and water treatment reagents, due to their ability to inhibit microbial contamination and biofilm formation [1]. Still, to the extent that the antimicrobial effect of zinc has been discovered, its impact on microbial communities and biofilm-forming bacteria, such as Escherichia coli and Enterococcus faecalis, in water systems remains to be thoroughly researched [2].

Biofilm formation in water supply systems is a highly significant public health and infrastructure management issue. Biofilms consist of complex microbial populations embedded in an extracellular polymeric substance (EPS) matrix, which allows bacteria to adhere to surfaces, thereby aiding their survival and contributing to antimicrobial resistance [3]. Biofilm growth in pipes can reduce water flow efficiency, increase energy consumption, and accelerate material degradation, ultimately leading to corrosion, pipe failure, and an increased risk of contamination. Additionally, biofilm-related bacteria exhibit heightened resistance to disinfectants and antibiotics, making them even more challenging to destroy [4]. In metal pipes, biofilm growth can initiate localized corrosion and compromise material strength. Recent studies have shown that certain metals, including zinc, influence biofilm dynamics by either inhibiting or stimulating bacterial attachment, depending on their chemical composition, concentration, and interaction with bacterial metabolism [5,6].

Zinc is an essential trace element for bacterial metabolism but exhibits antimicrobial properties at elevated concentrations by targeting several key biological processes. Specifically, zinc can inhibit metalloenzymes such as alcohol dehydrogenase and carbonic anhydrase, disrupting critical metabolic functions. It also displaces essential metal cofactors in enzymes, such as superoxide dismutase, impairing bacterial defenses against oxidative stress [6,7]. Furthermore, zinc interacts with membrane components such as phospholipids and membrane-bound proteins, leading to compromised membrane integrity, altered permeability, and ion imbalance, which together contribute to its bacteriostatic and bactericidal effects.

The mechanism of action of zinc-based antimicrobials is dependent on the solubility, dissociation properties, and ionic bioavailability of zinc compounds. For instance, zinc chloride is highly soluble and readily dissociates into Zn²⁺ ions, which induces increased antimicrobial activity. In contrast, zinc sulfate exhibits different solubility and reactivity that may alter its impact on bacterial biofilms [7]. Experiments showed that zinc nanoparticles can prevent bacteria adhesion and inhibit biofilm growth in Pseudomonas aeruginosa and Klebsiella pneumoniae [8]. However, the mechanisms by which zinc salts regulate biofilm formation in fecal indicator bacteria such as E. coli and E. faecalis have not been well elucidated. Evidence also shows that exposure to zinc may influence bacterial antibiotic resistance. Long-term exposure to zinc has been linked to multidrug resistance (MDR) in E. coli, particularly against aminoglycosides, cephalosporins, and sulfonamides, necessitating research on its involvement in antibiotic susceptibility [9]. Additionally, zinc regulates the response of bacteria to stress, likely modulating virulence factors and quorum-sensing gene expression that are responsible for antibiotic resistance and biofilm formation [10].

While there is a growing interest in the antimicrobial and biofilm-modulating characteristics of zinc, significant knowledge gaps remain concerning its effects on E. coli and E. faecalis biofilms in water distribution systems. This study evaluates the antibacterial effectiveness of zinc salts using minimum inhibitory concentration assays, biofilm formation tests, and antibiotic sensitivity profiling to assess their impact on these organisms. It aims to inform the potential design of antimicrobial strategies or additives for pipeline materials, to reduce microbial colonization.

Although this research does not fully elucidate molecular mechanisms, it provides essential phenotypic evidence of zinc’s influence on bacterial growth, biofilm development, and antibiotic susceptibility. These findings contribute to a broader understanding that can inform the future design of antimicrobial pipeline materials and support the development of biofilm control strategies aimed at enhancing water quality and infrastructure resilience.

2. Materials and Methods

2.1. Zinc Samples

The zinc samples used for testing are zinc sulfate heptahydrate (ZnSO₄·7H₂O, 424605000, Acros Organics, Geel, Belgium) and zinc chloride (ZnCl₂). The initial concentration was 10 mg/mL, representing the total mass concentration. This concentration was selected based on preliminary tests and prior studies examining bacterial responses to heavy metals. It provided a suitable baseline for preparing serial dilutions to assess both inhibitory and sub-inhibitory effects on bacterial growth and biofilm formation.

2.2. Microorganisms

The bacterial strains used were Escherichia coli ATCC 14169, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 19433, and Enterococcus faecalis ATCC 29212. The bacterial strains were cultured overnight at 37 °C on Mueller Hinton (MH) agar (Liofilchem Ltd., Roseto degli Abruzzi, Italy). The bacterial suspension used in the research was a 0.5 McFarland (1.5 × 10⁸ CFU/mL) inoculum prepared in MH broth (Liofilchem Ltd., Roseto degli Abruzzi, Italy). A total of 100 µL of the 0.5 McFarland inoculum was added to 9.9 mL of MH broth.

2.3. Tissue Culture Plate Method

The tissue culture plate method was used to determine the minimal inhibitory concentration (MIC). The negative controls used were MH broth. The positive controls (MH broth with bacteria) differed for each tested strain. Serial dilutions were made in MH broth in 96-well plates. The plates were inoculated with 20 µL of bacteria. The plates were incubated for 24 h at 37 °C. The optical density (OD) was measured in quadruplets at 595 nm using an ELISA plate reader (EPOCH, Agilent Technologies, Inc., Santa Clara, CA, USA).

2.4. Biofilm Formation Assaya

Zinc salts were used to evaluate their effect on biofilm formation for the tested bacteria. MH broth was used as a negative control, while the positive controls (MH broth with bacteria) were specific for each tested strain. The serial dilutions were made in MH broth in 96-well plates. The plates were incubated for 24 h at 37 °C. Afterward, they were washed, stained with 0.1% crystal violet, and treated with 96% ethanol. The OD was measured in quadruplets at 595 nm using the ELISA plate reader. The optical density cut-off value (ODc) was calculated with the following formula [11]:

ODc = average OD of negative control + 3 × standard deviation of negative control

(1)

The classification of biofilm formation is given in

Table 1. The classification of biofilm formation [11].

Formula	Biofilm Formation
OD ≤ ODc	Non-adherent
ODc < OD ≤ 2ODc	Weakly adherent
2ODc < OD ≤ 4ODc	Moderately adherent
4ODc < OD	Strongly adherent

.

2.5. Antibiotic Susceptibility Testing

The antibiotic susceptibility testing (AST) of the four reference bacterial strains was conducted in duplicate using the Kirby–Bauer disk diffusion method, as outlined in the Clinical and Laboratory Standards Institute (CLSI) guidelines [12]. The testing was conducted for the strains treated with the subminimum dose (the dilution below the determined MIC) of zinc salts. The McFarland of the subminimum dose was adjusted to 0.5, and the bacterial cultures were plated on MH agar plates. Each test was performed in triplicate. The antimicrobial disks (Liofilchem Ltd., Roseto degli Abruzzi, Italy) used were as follows: Doxycycline 30 µg (DXT 30), Amoxicillin 30 µg (AML 30), Mezlocillin (MEZ 75), Cefuroxime 30 µg (CXM 30), Ceftazidime 30 µg (CAZ 30), Ceftriaxone 30 µg (CRO 30), Ampicillin 2 µg (AMP 2), Amoxicillin and clavulanic acid 30 µg (AUG 30), Ceftazidime and clavulanic acid 40 µg (CAL 40), Ciprofloxacin 5 µg (CIP 5), Gentamicin 30 µg (CN 30), Gentamicin 10 µg (CN 10), Kanamycin 30 µg (K 30), Tobramycin 10 µg (TOB 10), and Tetracycline 30 µg (TE 30).

3. Results and Discussion

3.1. Minimum Inhibitory Concentration

Table 2. The minimum inhibitory concentration of zinc salts.

Bacterial Strain	MIC (mg/mL)
	ZnSO4 × 7H2O	ZnCl2
Escherichia coli ATCC 14169	5	10
Escherichia coli ATCC 25922	0.625	0.312
Enterococcus faecalis ATCC 19433	1.25	0.625
Enterococcus faecalis ATCC 29212	2.5	2.5

presents the MIC of two zinc salts against four bacterial strains.

Zinc chloride was more active than zinc sulfate heptahydrate against most bacterial strains, as evidenced by its lower MIC values. This can be attributed to its greater solubility and dissociation properties, leading to more bioavailable Zn²⁺ ions, which have been reported to exhibit antimicrobial activity through interference with bacterial cell membrane disruption and enzymatic processes.

The antimicrobial properties of zinc salts have been extensively explored in recent research. In one study, zinc sulfate heptahydrate suppressed multidrug-resistant bacteria, including E. coli and Enterobacter spp., with MIC values ranging from 10 mg/mL to 14 mg/mL depending on the bacterial species, in agreement with our finding for E. coli ATCC 14169 [13]. Additionally, the impact of Zn²⁺ ions on biofilms was established in a study that found that zinc sulfate caused considerable inhibition against E. coli biofilms at concentrations of only 0.005 M, with further inhibition observed at higher concentrations [14]. This supports the hypothesis that the antimicrobial activity of zinc salts is achieved through interference with biofilms, as well as their bacteriostatic potential.

In our present research, E. coli ATCC 14169 was the more resistant among the two, with MIC values of 5 mg/mL for zinc sulfate heptahydrate and 10 mg/mL for zinc chloride. The latter is compared with E. coli ATCC 25922, which was comparatively more susceptible. These differential sensitivities towards zinc are consistent with data from past literature, which shows that E. coli strains were similarly resistant due to genetic heterogeneity in efflux pumps and membrane permeability [15].

The results for E. faecalis indicate that bacterial species–specific factors, such as metal ion transporters and membrane permeability, underlie the demonstrated MIC differences. Studies on E. faecalis have confirmed that ZnO nanoparticles exhibit more antimicrobial action against this species than against other Gram-negative bacteria, again reinforcing the effect of the structure of bacterial cell walls on susceptibility to zinc ions [16].

The present work also supports previous reports that zinc chloride exhibits stronger antimicrobial activity than zinc sulfate heptahydrate. This has been validated by research on various zinc salts, where zinc chloride exhibited enhanced antimicrobial activity against Streptococcus pyogenes and E. coli due to its more effective ionic dissociation, resulting in the higher bioavailability of zinc [14]. Nonetheless, based on other research, biofilm aging, metal sequestration mechanisms, and inhibition by enzymes may regulate the antibacterial effect of zinc salts [5].

3.2. The Effect on the Planktonic Growth

The inhibition of zinc sulfate heptahydrate (Figure 1) and zinc chloride (Figure 2) on planktonic bacterial growth provides insight into their dose-dependent antimicrobial action against E. coli and E. faecalis. The results reveal differential bacterial responses, with the higher concentrations of zinc (10 mg/mL and 5 mg/mL) exhibiting the most significant inhibition, as indicated by reduced optical density (OD) values for all bacterial strains.

Our findings align with those of previous research, which demonstrate the growth-inhibitory properties of zinc salts against bacteria. In one paper, it was noted that zinc aspartate in high doses (2500 µg/mL) had a bactericidal effect on E. coli and Staphylococcus aureus biofilms, which supports the observation that the antimicrobial property of zinc is dose-dependent [17].

Surprisingly, zinc chloride was more inhibitory at lower concentrations (0.625–0.156 mg/mL) than zinc sulfate heptahydrate, contrasting with another study’s findings, where zinc sulfate was the most toxic zinc compound to Bacillus subtilis, Lactococcus lactis, and Saccharomyces cerevisiae, with higher concentrations required for significant inhibition [18]. Danilova et al. also found that E. coli biofilms were less sensitive to zinc chloride at low concentrations but were effectively inhibited at high concentrations [14]. Previous studies show that genetic variation between E. coli strains affects their resistance to zinc ions [19].

E. faecalis strains exhibited slow growth at low zinc concentrations, suggesting moderate resistance compared to E. coli. This finding concurs with research indicating that E. faecalis exhibited intermediate susceptibility to ZnO nanoparticles, and planktonic growth inhibition depended on zinc ion release [20].

Zinc chloride’s higher antimicrobial activity in mid-range concentrations (1.25–0.312 mg/mL) also supports the role of higher Zn²⁺ bioavailability in bacterial inhibition. Compared to zinc sulfate heptahydrate, zinc chloride releases Zn²⁺ more readily due to greater solubility, leading to higher uptake and toxicity against bacteria. The same was noted when researching zinc-based nanoparticles, where higher ionization led to greater antibacterial activity [21].

3.3. The Effect on the Biofilm Formation

The action of zinc sulfate heptahydrate (Figure 3) and zinc chloride (Figure 4) on E. coli and E. faecalis biofilm growth is relevant to the antimicrobial activity and biofilm-modulating efficacy of zinc salts. It was found that whereas biofilm formation was inhibited in high concentrations of zinc (≥2.5 mg/mL), sub-inhibitory concentrations (0.156–0.019 mg/mL) increased the yield of the biofilm, suggesting bacterial stress response.

Our findings are consistent with previous work that has found that high concentrations of zinc salts inhibit biofilm formation. Buzza et al. found that the biofilm formation of Streptococcus mutans was inhibited by zinc acetate, which increased the permeability of biofilms at doses above 0.3 mg/mL, thereby validating the assumption that Zn²⁺ ions can disrupt the integrity of the biofilm at bactericidal doses [5]. However, under sub-inhibitory concentrations (0.156–0.019 mg/mL), biofilm development was enhanced in E. coli ATCC 14169, E. faecalis ATCC 19433, and E. faecalis ATCC 29212, indicating that biofilm development is a survival mechanism against low zinc levels. The same has been observed in P. aeruginosa and S. aureus, where sub-lethal zinc concentrations triggered biofilm development under metal stress [1]. Similarly, Mahamuni-Badiger et al. demonstrated that exposure to low doses of zinc promotes biofilm development in bacteria associated with medical devices, highlighting the bimodal nature of zinc’s modulation of biofilms [22].

Zinc salts exhibit antimicrobial action through the disruption of membrane integrity, inhibition of enzymes, and inhibition of oxidative stress by Zn²⁺ ions. At high concentrations, low inhibitory concentrations may facilitate the development of biofilms and resistance. Antimicrobial peptides (AMPs) possess broad-spectrum and often targeted action, with less potential for resistance due to their rapid, membrane-targeted mechanism. A recent survey identified over 300 novel AMPs from marine biofilm bacteria, with most exhibiting vigorous activity against drug-resistant microorganisms [23]. The discoveries suggest that AMPs can be promising alternatives or adjuncts to zinc, especially in applications where specificity is required and little resistance potential exists.

Zinc chloride inhibited biofilm development more effectively at higher concentrations (10–2.5 mg/mL) but elicited stronger biofilm reactions at low concentrations. E. coli ATCC 14169 produced more biofilm at 0.156 mg/mL and attained a peak at 0.078 mg/mL, demonstrating that zinc chloride stress at low concentrations triggers biofilm production. E. coli ATCC 25922 produced the most significant increase at 0.312 mg/mL, supporting enhanced protective biofilm production under zinc chloride stress. These findings align with those of Bianchini Fulindi et al., who indicated that zinc sulfide and zinc oxide nanoparticles significantly inhibited the growth of Klebsiella oxytoca and P. aeruginosa biofilms and induced protective biofilm responses at sub-lethal concentrations [24].

Zinc sulfate heptahydrate induced a slower-growing biofilm response in sub-inhibitory concentrations, where E. faecalis ATCC 19433 and E. faecalis ATCC 29212 showed greater biofilm production at 0.019 mg/mL. This means that Gram-positive E. faecalis strains employ biofilm development as a survival strategy for low Zn²⁺ stress. Yi et al. demonstrated that zinc nitrate microneedles successfully destroyed mature biofilms but also caused transient biofilm formation at low zinc concentrations [25].

Zinc at sub-inhibitory levels appears to control bacterial biofilm formation by disrupting quorum sensing and EPS synthesis. Zinc was found to suppress quorum sensing at high concentrations but trigger biofilm signaling at low concentrations [3]. Sub-lethal exposure to Zn²⁺ may initiate a stress response that results in the overproduction of the EPS, leading to biofilm development. Similar effects were observed in Proteus mirabilis, where zinc oxide nanoparticles suppressed biofilm gene expression at inhibitory concentrations but stimulated protective biofilm responses at sub-inhibitory doses [26].

3.4. Antibiotic Susceptibility Testing Results

The antibiotic sensitivity data presented in

Table 3. The antibiotic susceptibility for E. coli.

ABG	E. coli ATCC 14169			E. coli ATCC 25922
	PC	ZnSO4 × 7H2O	ZnCl2	PC	ZnSO4 × 7H2O	ZnCl2
DXT 30	15.00 ± 0.00	16.50 ± 0.71	15.50 ± 0.71	21.00 ± 0.00	20.50 ± 0.71	21.00 ± 0.00
AML 30	20.00 ± 0.00	22.00 ± 0.00	21.00 ± 0.00	21.00 ± 0.00	20.00 ± 1.41	18.50 ± 0.71
MEZ 75	21.00 ± 0.00	19.00 ± 0.00	21.50 ± 0.71	26.00 ± 1.41	30.00 ± 3.54	25.50 ± 0.71
CXM 30	19.00 ± 0.00	20.50 ± 0.71	18.00 ± 0.00	24.00 ± 0.00	24.00 ± 0.00	24.00 ± 0.00
CAZ 30	22.50 ± 0.71	22.00 ± 0.00	22.50 ± 0.71	28.00 ± 0.00	28.00 ± 0.00	29.00 ± 0.00
CRO 30	28.00 ± 0.00	30.00 ± 0.00	27.00 ± 0.00	32.00 ± 0.00	32.00 ± 0.00	32.50 ± 0.71
AMP 2	R	R	R	R	R	R
AUG 30	20.00 ± 0.00	21.00 ± 0.00	19.00 ± 1.41	19.00 ± 1.41	21.00 ± 0.00	17.50 ± 0.71
CAL 40	25.00 ± 0.00	24.50 ± 0.71	25.00 ± 0.00	28.50 ± 0.71	29.50 ± 0.71	28.50 ± 0.71
CIP 5	31.00 ± 1.41	33.00 ± 0.00	31.00 ± 1.41	35.50 ± 0.71	34.00 ± 0.00	34.00 ± 0.00
CN 30	21.50 ± 0.71	22.00 ± 0.00	22.00 ± 0.00	25.50 ± 0.71	25.50 ± 0.71	25.50 ± 0.71
CN 10	19.50 ± 0.71	20.50 ± 0.71	20.00 ± 0.00	22.00 ± 0.00	22.00 ± 0.00	22.00 ± 0.00
K 30	18.00 ± 0.00	19.00 ± 0.00	17.00 ± 0.00	21.00 ± 0.00	21.50 ± 0.71	22.00 ± 0.00
TOB 10	18.50 ± 0.71	20.00 ± 0.00	18.50 ± 0.71	21.00 ± 0.00	19.50 ± 0.71	21.00 ± 0.00
TE 30	15.00 ± 0.00	13.00 ± 0.00	15.50 ± 0.71	19.50 ± 0.71	20.00 ± 0.00	18.50 ± 0.71

PC—positive control, nontreated strains; R—resistant. The data represent zones of inhibition in mm.

and

Table 4. The antibiotic susceptibility for E. faecalis.

ABG	E. faecalis ATCC 19433			E. faecalis ATCC 29212
	PC	ZnSO4 × 7H2O	ZnCl2	PC	ZnSO4 × 7H2O	ZnCl2
DXT 30	15.50 ± 0.71	17.50 ± 0.71	12.50 ± 0.71	11.00 ± 0.00	13.50 ± 0.71	15.00 ± 0.00
AML 30	20.00 ± 0.00	21.50 ± 0.71	19.00 ± 1.41	20.00 ± 0.00	27.00 ± 0.00	27.00 ± 0.00
MEZ 75	20.50 ± 0.71	23.50 ± 0.71	21.50 ± 0.71	26.00 ± 1.41	25.00 ± 0.00	28.50 ± 0.71
CXM 30	17.50 ± 0.71	19.50 ± 0.71	17.00 ± 0.00	16.00 ± 1.41	R	R
CAZ 30	22.00 ± 0.00	24.00 ± 0.00	22.00 ± 0.00	10.50 ± 0.71	R	R
CRO 30	27.50 ± 0.71	29.00 ± 0.00	26.00 ± 1.41	20.00 ± 0.00	R	R
AMP 2	R	R	R	10.00 ± 0.00	14.50 ± 0.71	15.50 ± 0.71
AUG 30	19.00 ± 0.00	18.50 ± 0.71	18.00 ± 0.00	24.00 ± 0.00	26.00 ± 0.00	25.00 ± 0.00
CAL 40	23.50 ± 0.71	25.00 ± 0.00	16.00 ± 1.41	12.00 ± 0.00	R	13.50 ± 0.71
CIP 5	31.50 ± 0.71	30.00 ± 0.00	27.50 ± 0.71	20.00 ± 0.00	20.00 ± 0.00	21.00 ± 1.41
CN 30	21.50 ± 0.71	21.50 ± 0.71	20.00 ± 0.00	17.50 ± 0.71	15.00 ± 0.00	20.00 ± 0.00
CN 10	19.50 ± 0.71	19.50 ± 0.71	17.00 ± 0.00	11.50 ± 0.71	10.50 ± 0.71	17.00 ± 0.00
K 30	18.50 ± 0.71	17.50 ± 0.71	16.50 ± 0.71	25.50 ± 0.71	10.00 ± 0.00	12.00 ± 0.00
TOB 10	18.00 ± 0.00	19.00 ± 0.00	19.50 ± 0.71	26.00 ± 1.41	10.00 ± 0.00	14.00 ± 0.00
TE 30	12.00 ± 0.00	13.00 ± 0.00	16.00 ± 1.41	11.50 ± 0.71	12.00 ± 0.00	14.00 ± 0.00

PC—positive control, nontreated strains; R—resistant. The data represent zones of inhibition in mm.

hold significant information regarding the influence of zinc salts on the antibiotic resistance of E. coli and E. faecalis strains. The results suggest that treating bacteria with zinc sulfate heptahydrate and zinc chloride alters bacterial sensitivity to various antibiotics, with notable differences observed for different bacterial species and antimicrobials.

Our results indicate that zinc chloride tends to enhance susceptibility to a range of antibiotics in E. coli, particularly ciprofloxacin, amoxicillin, and mezlocillin. In contrast, susceptibility in E. faecalis to cephalosporins is reduced, suggesting a strain-specific action.

The impact of zinc salts on antibiotic resistance has been investigated increasingly. Adekanmbi et al. investigated zinc-resistant E. coli isolates of environmental origin that were zntA-positive. They found that exposure to zinc increased resistance to ampicillin (92.6%) and amoxicillin-clavulanate (68.5%), but susceptibility to ciprofloxacin was not significantly affected [27]. This contrasts with our findings, in which zinc chloride enhanced susceptibility to ciprofloxacin in E. coli, showing strain-specific interaction with zinc.

Nechifor et al. demonstrated the role of zinc in altering antibiotic efficacy, with zinc enhancing bacterial sensitivity to β-lactam antibiotics, which corroborates our findings, where treatment with zinc chloride increased the zone of inhibition of amoxicillin in E. faecalis ATCC 29212 [28]. On the other hand, E. faecalis had decreased susceptibility to ceftazidime and ceftriaxone, supporting the theory that exposure to zinc may enhance β-lactam resistance in some species. An increase in the ciprofloxacin zone of inhibition after treatment with zinc chloride suggests a possible synergistic effect, as previously observed. Andrews et al. reported that zinc-activated avobenzone possessed potent antibacterial activity against MRSA, highlighting the capacity of zinc to enhance fluoroquinolone activity [29].

Zinc exposure has also been reported to disrupt bacterial membrane integrity, which affects antibiotic uptake. This is also reflected in studies that suggest that zinc oxide nanoparticles can enhance the penetration of antibiotics, particularly aminoglycosides such as gentamicin [9]. This could explain the slight enhancement of gentamicin activity following zinc chloride treatment in our study. Exposure to zinc has been found to induce efflux pumps, leading to increased resistance to certain antibiotics [30]. Zinc exposure is linked to oxidative stress, which can either compromise bacterial defenses (sensitizing bacteria to antibiotics) or induce protective responses (increasing resistance). This was demonstrated by Ye et al., who provided evidence that iron and zinc ions regulate efflux pumps and biofilm formation, thereby influencing multidrug resistance [31].

This work is founded on our earlier work on the effects of iron salts on microbial populations under such pipeline conditions [32]. There are interconnected trends in the two studies: large amounts of metals suppress biofilm development and microbial growth. At the same time, lower concentrations trigger stress-adaptive mechanisms in E. coli and E. faecalis, including a change in antibiotic resistance. These findings suggest the need for carefully optimized dosing regimens to harness the antimicrobial action of metal ions without promoting resistance or the formation of biofilms.

Although our study did not directly test zinc-integrated pipeline material, the anti-biofilm and antimicrobial activities observed here suggest that zinc salts—especially zinc chloride, due to its higher bioavailability—could be considered a candidate coating or additive for pipeline infrastructure. Zinc addition to polymers or metal alloys would be worth testing in the future under simulated flow conditions to establish practical feasibility.

4. Conclusions

This research identifies a dual function for zinc salts, which act as both antimicrobial compounds and modulators of biofilms. Strain differences in zinc tolerance nonetheless indicate that intrinsic resistance mechanisms in bacteria, such as efflux pumps and metal sequestration activities, contribute profoundly to susceptibility to zinc. The findings confirm that high concentrations of zinc chloride and zinc sulfate heptahydrate are effective in inhibiting biofilm formation. In contrast, sub-inhibitory concentrations induce biofilm production, particularly in E. coli and E. faecalis, as a survival mechanism. This finding aligns with current research indicating that exposure to sub-lethal metal concentrations can enhance bacterial resistance by promoting biofilm formation and altering antibiotic susceptibility. Based on these findings, zinc antimicrobial strategies should be carefully optimized to prevent the unintended emergence of resistance in clinical and industrial applications.

One limitation of this study is the use of well-characterized ATCC reference strains, which, while consistent and widely used in laboratory research, may not fully represent the genetic and phenotypic diversity of environmental isolates found in actual pipeline systems. These variables can affect biofilm formation, metal–microbe interactions, and antimicrobial efficacy in ways that experiments cannot replicate. Additionally, the anti-biofilm activity was assessed using crystal violet staining and absorbance measurements, which quantify total biofilm biomass but do not differentiate between viable and non-viable cells. Furthermore, these methods do not provide threshold concentrations for biofilm inhibition or eradication. Established biofilm-specific metrics, such as the Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC), were not employed due to methodological constraints; however, they should be considered in future studies for a more accurate and clinically relevant evaluation.

Furthermore, the study did not include microscopic observations to visualize biofilm architecture or confirm zinc’s structural effects on biofilm formation, which limits the interpretation of morphological changes. Despite these limitations, the findings highlight the significant role of zinc salts in influencing microbial behavior, underscoring the need for effective biofilm control within water infrastructure.

To build on this research, future efforts should focus on investigating zinc–bacteria interactions under dynamic, realistic conditions, with representative flow regimes, mixed-species biofilms, and environmental stress. In addition, investigating new materials for pipelines and emerging coatings that achieve a balance of biofilm repression, durability, and cost may provide promising solutions for maintaining water system performance with minimal risk of microbial contamination.