Control of Microbial Contamination on Dairy Farms Using Multicomponent Disinfectants: An In Vitro Study

1. Introduction

Destruction of microorganisms in the environment is of great importance for preventing the spread of infectious diseases. The emergence of microorganisms that are tolerant to disinfectants is a concern for product manufacturers, veterinarians, and human health practitioners. Resistance to aldehyde-based and quaternary ammonium compounds (QAC) biocides has been identified in Campylobacter jejuni, Enterococcus faecium, Escherichia coli, Staphylococcus aureus [1]. Aldehyde and QAC-based disinfectants are considered to be quite active biocides with a broad spectrum of action. They should be applied at appropriate effective concentrationsand to determine the sensitivity of microorganisms in advance. In addition, to prevent the development of tolerance in microorganisms, it is necessary to periodically rotate disinfectants.

Also, common biocides for disinfection in livestock facilities are chlorine-based agents, which have a broad spectrum of antimicrobial activity [2], however, the resistance of spore-forming bacteria to chlorine has been proven [3]. Additionally, chlorine disinfection has been linked to increased bacterial resistance to antibiotics [4].

Previous studies have demonstrated that the use of sublethal disinfectant concentrations of disinfectants to kill bacteria can lead to their survival and further development of adaptive properties, such as the formation of biofilms. Biofilms make it difficult for active molecules of disinfectants to penetrate and protect microorganisms from their harmful effects. Accordingly, the use of an iodine-based product on the farm in a concentration (1:50) rather than the recommended (1:90) resulted in residual Salmonella spp. contamination after disinfection [5].

A study of iodine disinfection of Casper fish embryo surfaces shows that its use is effective at concentrations to kill Mycobacterium (12.5–25 ppm) compared to NaOCl (50–100 ppm). Furthermore, it has been proven to be non-toxic and embryo survival has resulted in >50% survival compared to NaOCl [6].

Kim et al. [7] evaluated the efficacy of a combination disinfectant based on 70% isopropyl alcohol and 10% povidone-iodine. The combination of isopropyl alcohol and povidone-iodine had a superior effect compared to disinfection with a single agent. The authors also note that the combination of several active components provides a broader spectrum of antimicrobial activity.

The use of copper iodine complex for hand sanitizing of healthcare workers has demonstrated effectiveness against SARS-CoV-2 [8]. However, the study does not report the possibility of using the agent against other viruses, bacteria and microscopic fungi.

The aim of this study was to investigate the in vitro sensitivity of opportunistic microorganisms isolated from dairy livestock facilitiesto multicomponent disinfectants based on various active substances, to establish their effective working concentrations in vitro, and to scientifically substantiate the possibility of using these disinfectants in rotational disinfection schemes for dairy farms. The study also aimed to form a scientific basis for the implementation of disinfectant rotation programs aimed at increasing the effectiveness of preventive measures and reducing the risk of developing resistance of microorganisms to disinfectants.

2. Materials and Methods

2.1. Material Collection

Environmental microbial samples were collected from dairy cow housing facilities on seven dairy farms in the Sumy region, Ukraine. The seven dairy farms included in the study were selected according to predefined housing criteria. Only farms operating a free-stall housing system were enrolled, allowing cows unrestricted movement within the pens. Each pen accommodated approximately 40–50 cows, and each barn contained two to four pens depending on the facility size. These criteria were used to ensure comparable housing and management conditions across all farms. The animals had to be kept loose and able to move freely around the pen. Each pen accommodates 40-50 cows. Depending on the size of the building, there can be 2-4 pens in a cowshed. Surface samples were collected using sterile eSwab™ collection systems (Copan, USA). To avoid interference from recently applied disinfectants, no chemical disinfection had been performed in the barns for at least 10 days before sampling; only routine mechanical cleaning was carried out. Samples were collected from surfaces with direct animal contact (floors, resting areas, feeders, drinkers, and pen partitions) and from surfaces without direct animal contact (walls, door handles, latches, ceilings, and equipment). One swab sample was collected from each sampling site. The obtained samples were transported to the laboratory in an insulated container within 3–4 h of collection. A total of 98 samples were examined. The research was conducted between January and February 2026.

2.2. Preparation of Disinfectant Test Solutions

Three commercial disinfectants (PE Kronos Agro, Ukraine) were evaluated to determine the minimum cidal concentration (MCC). Disinfectant I consisted of 5% crystalline iodine, 2.5% potassium iodide, 1.2% lactic acid, and excipients including glycerol, isopropyl alcohol, ethoxylated alcohol, and water. The composition of disinfectant II included 13% glutaraldehyde, 4% glyoxal, 11% formaldehyde, 6% quaternary ammonium compounds (QACs), 1.5% triamine, 8–10% fogging agents (triethylene glycol and glycerin), and water up to 100%. Disinfectant III had the following composition: 8% glutaraldehyde, 8% glyoxal, 15% benzalkonium chloride, 5% dodecyldimethylammonium chloride, 0.5% triethylene glycol and glycerin as fogging agents, and water up to 100%. For in vitro studies, appropriate disinfectant solutions were prepared in clean tap water. Disinfectant I was used at concentrations of 5, 3, 1.5, and 0.5%, and disinfectants II and III at concentrations of 1, 0.5, 0.3, and 0.1%. Freshly prepared disinfectant solutions were used immediately after preparation.

2.3. Microbial Screening

Microbiological analyses were performed at the Microbiological Laboratory of the Medical Institute of Sumy State University and the Scientific Laboratory of PCR diagnostics of Sumy National Agrarian University. For isolation of a pure culture, the obtained samples were inoculated using the streak plate method on differential diagnostic and selective media: Endo agar (EA), yolk-salt agar (YSA), blood agar (BA), chocolate agar (CHA) and Sabouraud agar (SA) manufactured by Pharmaktiv LLC (Ukraine), as well as Pseudomonas agar (PSA) from HiMedia (India). Inoculated Petri dishes were incubated aerobically for 24-48 hours at 37 ° C. Anaerobic bacteria were cultured on blood agar supplemented with hemin (5 mg/L) and vitamin K₁ (1 mg/L). The cultures were incubated under anaerobic conditions in a microaerostat at 37 °C for 48–72 hours. The isolated microorganisms were identified based on colony morphology, Gram-staining characteristics, culture characteristics, and biochemical profiles [9].

2.4. Determination of the Cidal Activity of Disinfectants

The effective bactericidal concentrations of disinfectants against selected microbial species were determined using a culture-based approach followed by subculturing onto solid nutrient media [10]. The experiments were performed using a broth microdilution assay. Each well contained 100 μL of Mueller–Hinton broth, 20 μL of freshly prepared disinfectant solution, and 2 μL of microbial suspension with a concentration approximately 1×10⁶ CFU/mL, resulting in a final volume of 122 μL. The concentrations presented in Table 1 correspond to the final disinfectant concentrations in the wells after dilution in the total assay volume (122 μL). The microplate was incubated at 37°C. After 24 hours incubation, aliquots from each well were streaked onto Mueller-Hinton medium (Farmaktiv LLC, Ukraine) and incubated for 24 hours at 37°C to assess the cidal activity of each concentration of disinfectant. The study was performed in duplicate.

2.5. Statistical Analysis

All experiments were performed in duplicate. Microbial growth was evaluated as a binary outcome (growth/no growth). The MCC was defined as the lowest disinfectant concentration resulting in complete absence of growth in all replicates. Data were summarized descriptively and presented as growth/no-growth observations.

3. Results

3.1. Microbial Profile of Livestock Facilities of Dairy Farms

According to the conducted studies, the following types of microorganisms were isolated in samples obtained from the surfaces of livestock facilities: S. aureus, E. coli lac+ and lac-, Pseudomonas aeruginosa, Bacteroides flagilis, Klebsiella spp., Proteus spp., Prevotella spp., Acinetobacter spp., and also mold fungi and Candida non-albicans.

Notably, the microbial profile differed in different locations of the livestock facilities, namely: in areas with high humidity (drinking troughs, manure accumulation areas, floors), gram-negative microorganisms prevailed, in particular E. coli, P. aeruginosa, Klebsiella spp. and representatives of species Proteus and Acinetobacter, as well as anaerobic bacteria (Bacteroides, Prevotella).

Instead, on drier surfaces (walls, fences, equipment) gram-positive cocci were more frequently detected, primarily S. aureus. In areas with insufficient ventilation and increased organic pollution, the presence of fungal microflora was noted, in particular mold fungi and yeasts of the Candida (non-albicans) species.

Such differences indicate the influence of microclimatic conditions, humidity levels, organic load, and the intensity of animal contact with surfaces on the formation of the local microbial profile within livestock facilities.

3.2. MCC of Disinfectants

Determination of the MCC using the classical method allowed establishing effective concentrations of disinfectants against selected species of microorganisms.

Analysis of the obtained data indicates different sensitivity in vitro of the studied microorganisms to iodine-containing disinfectant depending on its concentration (Table 1). It was found that the concentration of 0.5% was not effective enough, since growth was observed for all studied microorganisms. When the concentration was increased to 1.5%, partial inhibition of microflora was observed, in particular E. coli lac-. This indicates its low resistance compared to other studied microorganisms. A 3% concentration of disinfectant I produced a cidal effect against most microorganisms, including S. aureus, E. coli lac+, P. aeruginosa, Proteus spp., Acinetobacter spp., Prevotella spp., Klebsiella spp., as well as fungal microflora (mold fungi and Candida non-albicans). The results suggest lower susceptibility of B. fragilis to the iodine-based disinfectant under the experimental conditions used in this study.

Complete growth inhibition of all studied microorganisms was observed at a concentration of disinfectant I equal to 5%, which indicates the achievement of the MCC for this disinfectant at this level.

Therefore, the results obtained indicate that the effectiveness of iodine-containing disinfectant has a clear dose dependence. The most resistant among the studied microorganisms was B. fragilis, while E. coli lac- was characterized by the highest sensitivity. It is practically significant that to ensure guaranteed bactericidal action against mixed microflora of livestock facilities, it is advisable to use a concentration of not less than 5%.

The results shown in Table 2 indicate a pronounced variability in the sensitivity of the studied microorganisms to a disinfectant based on aldehydes and QAC, depending on the concentration.

It was found that at a concentration of 0.1%, the disinfectant did not provide a cidal effect on most microorganisms, as evidenced by the presence of growth (S. aureus, E. coli lac+, E. coli lac-, P. aeruginosa, B. fragilis, Proteus spp., as well as fungal microflora). At the same time, Acinetobacter spp., Prevotella spp. and Klebsiella spp. did not show growth even at this concentration, which indicates their high sensitivity.

At a concentration of 0.3% disinfectant II, a significant increase in efficiency was observed: the absence of growth was characteristic of S. aureus, E. coli lac+ and P. aeruginosa. However, E. coli lac-, B. fragilis and Proteus spp. retained viability, indicating their greater resistance. A similar trend was observed at a concentration of 0.5%.

The highest concentration (1%) provided a cidal effect on most of the microorganisms studied. However, even under these conditions, S. aureus and E. coli lac+ demonstrated growth, which may indicate their increased resistance to the components of this disinfectant. At the same time, for E. coli lac-, P. aeruginosa, B. fragilis and Proteus spp. at this concentration, a complete lack of growth was observed.

Fungal microflora was characterized by relative resistance at low and medium concentrations (0.1–0.5%), but their growth was completely inhibited at a 1% concentration of disinfectant II.

It was established that the studied disinfectant has a concentration-dependent cidal activity. The most sensitive were Acinetobacter spp., Prevotella spp. and Klebsiella spp., while increased resistance was shown by S. aureus and E. coli lac+. Under the experimental conditions of this in vitro study, a disinfectant concentration of at least 1% was required to achieve complete growth inhibition of the tested microorganisms. Further studies under field conditions are needed to confirm the effectiveness of this concentration in dairy farm environments.

The results obtained (Table 3) indicate different sensitivity of the studied microorganisms to the disinfectant III (aldehyde- and QAC-based) depending on the concentration. The least effective concentration was 0.1%, at which the growth of most microorganisms was preserved, in particular S. aureus, E. coli (lac+ and lac-), B. fragilis, Klebsiella spp., as well as fungi. At the same time, some bacteria P. aeruginosa and representatives of the species Proteus spp., Acinetobacter spp. and Prevotella spp. did not show growth at this concentration, which indicates their high sensitivity.

When the concentration of disinfectant III is increased to 0.3%, a significant decrease in growth is observed: E. coli lac- and Klebsiella spp. are inhibited, but S. aureus, E. coli lac+, B. fragilis and fungi retain their viability. The concentration of 0.5% is critical for most of the studied microorganisms: the growth of E. coli (both variants), B. fragilis, fungi, as well as most gram-negative bacteria is completely inhibited. At the same time, S. aureus still retains growth, which indicates its increased resistance.

After increasing the concentration of disinfectant III to 1%, there is a complete absence of growth of almost all microorganisms, including S. aureus, which allows us to consider it as an effective bactericidal and fungicidal concentration. The exception is Klebsiella spp., for which re-growth is recorded in Table 3. Therefore, we consider this finding to be most likely related to experimental variability rather than a true decrease in disinfectant activity.

4. Discussion

The concentration of microorganisms in a cowshed depends on environmental conditions (temperature, humidity, air velocity, light), season, and number of animals [11].

The present study revealed uneven localization of microorganisms inside the livestock facilities of dairy farms. Anaerobes (B. fragilis, Prevotella spp.), Gram-negative microorganisms – E. coli, P. aeruginosa, Klebsiella spp. and Proteus spp., as well as Acinetobacter spp. were isolated in places of constant contact with animals (floor, drinkers). Consequently, in previous studies [12] it was reported that E. coli, Enterobacter aerogenes and Proteus mirabilis were isolated from the floor in 36% of samples, and S. aureus – in 50%. It is also known [13] that Staphylococcus spp. were isolated to a greater extent in facilities for different animal species. On Slovak dairy farms [14] coliform bacteria were isolated in the air of cattle in concentrations from 2.18 log10 CFU/ml to 3.34 log10 CFU/ml; and for mold, concentrations ranged from 3.00 log10 CFU/mL to 4.57 log10 CFU/mL.

It is very important to consider the management in the cowshed when choosing a disinfectant [15,16]. The treatment of the main place where cows stay, namely the walking area, the feed table (drinkers, feeders) must be carried out regularly at least once a week to prevent the spread of pathogens throughout the farm [17]. Dairy cows move to the milking parlors two to three times and during this period it is convenient to carry out current disinfection. As researchers note [18], the problem is non-compliance with hygiene and lack of disinfection. However, the use of a single-component disinfectant cannot ensure the destruction of all microorganisms, so complex disinfectants are used on farms to ensure the widest possible spectrum of action. Therefore, multicomponent disinfectants with different classes of active substances were selected for our study. Studies [19] indicate that frequent use of the same biocidal products can lead to the development of tolerance-increasing mechanisms in bacteria, such as the formation of biofilms.

A study of the MCC of an iodine-containing disinfectant showed that a concentration of 0.5% was not effective against all bacterial isolates. Increasing the concentration to 1.5% showed biocidal activity against E. coli lac-. Previous studies [20,21] confirm the effectiveness of using iodine as a disinfectant for the destruction of microorganisms.

The results of our studies are consistent with the data of Lahnsteiner and Dunser [22] on the bactericidal efficacy of an iodine-containing disinfectant. In addition, the authors report a long-lasting bactericidal effect.

A 3% concentration of iodine-based disinfectant was cidal against all bacterial and fungal isolates except B. fragilis in duplicate. The MCC of iodine-based disinfectant is 5% for effective control of bacteria and microscopic fungi in a cowshed. The results of our studies are consistent with previously published works [23,24,25]. The sensitivity of microorganisms to iodine-containing agents varied between organisms and was directly dependent on its concentration. Based on the results of our experiment, we propose to use an iodine-based disinfectant for routine disinfection in the barn in places where animals are constantly kept.

In vitro studies of an aldehyde+QAC disinfectant showed that a concentration of 0.1% was effective against Acinetobacter spp., Prevotella spp. and Klebsiella spp., which is 27% of the total number of isolated opportunistic microorganisms. In our study, S. aureus, E. coli lac+ and P. aeruginosa showed sensitivity to concentrations of 0.3-0.5%. The results obtained are consistent with the data of Schug et al. [26].

Previous studies have shown that the minimum inhibitory concentration of QAC for C. albicans and Candida auris is 16–32 mg/l [27]. Therefore, when performing disinfection with QAC-based agents, it is necessary to adhere to effective cidal concentrations.

Increasing the concentration of disinfectant II by two times to 1% contributed to an increase in sensitivity in all tested isolates, except for S. aureus and E. coli lac+. That is, the bactericidal efficiency of the disinfectant at a concentration of 1% was 80%, which is a sufficient indicator. It is also important that at a concentration of 0.1–0.5%, disinfectant II has a pronounced fungicidal effect. Therefore, for use on farms during preventive and final disinfection, it is advisable to use a concentration not lower than 1%.

Previous studies [28] have shown that it is important to establish a minimum inhibitory concentration of the biocide for wild isolates of microorganisms. Determination of the biocidal efficacy of a disinfectant III, which was also based on aldehydes and QAC, showed that a concentration of 0.1% was effective against 36% of the tested microorganisms. Increasing the biocide concentration to 0.3% showed an efficacy of 54%. A concentration of disinfectant III of 0.5% killed 90% of all isolates except S. aureus. All microorganisms were susceptible to a 1% concentration of disinfectant III, except for Klebsiella spp. (±), which was not susceptible in one of the two replicates.

Researchers [29] have reported that disinfectants can also increase cross-resistance to antibiotics. Kampf [30] reported that he did not detect any cross-resistance to antibiotics after low-level exposure to QAC.

Overall, aldehyde- and QAC-based disinfectants demonstrated the broadest antimicrobial spectrum, whereas iodine-based formulations required higher concentrations to achieve complete microbial inactivation. However, all tested disinfectants showed concentration-dependent efficacy. These findings may support evidence-based selection of disinfectants for routine biosecurity programs on dairy farms.

5. Conclusions

This in vitro study determined the minimum cidal concentrations (MCCs) of three disinfectants with different active ingredients against opportunistic microorganisms isolated from dairy livestock facilities. The iodine-based disinfectant demonstrated effective antimicrobial activity at a concentration of 5%, making it suitable for routine disinfection in the presence of animals. The aldehyde- and quaternary ammonium compound (QAC)-based disinfectants showed broad-spectrum antimicrobial activity at a concentration of 1%, supporting their use for preventive and terminal disinfection of livestock facilities. These findings provide evidence-based guidance for selecting effective disinfectants to reduce microbial contamination in dairy production systems.

Limitations. The antimicrobial activity of the disinfectants was evaluated only under controlled in vitro conditions using planktonic bacterial and fungal isolates and were not evaluated using standardized AOAC or EN efficacy protocols. However, the use of wild field isolates, rather than laboratory reference strains, increases the practical relevance of the findings by better reflecting the microbial populations encountered on dairy farms. The efficacy of the disinfectants against microbial biofilms, which are common on livestock farm surfaces and may exhibit increased tolerance to biocides, was not investigated and represents an important direction for future research. The influence of organic matter (e.g., manure, bedding material, and milk residues), which may reduce disinfectant activity under farm conditions, was not assessed. The disinfectants were not evaluated under real farm environmental conditions, where factors such as surface type, temperature, humidity, and application method may affect their performance. Our study did not include an assessment of virucidal activity. Therefore, further in vivo and field-based studies are warranted to validate the efficacy of these disinfectants under practical farm conditions, including their activity against biofilms, in the presence of organic contamination, and against viral pathogens.