Antibacterial and Antifungal Effects of Chemical Additives Used in Poultry Production: An In Vitro Minimum Inhibitory Concentration Study

Abstract

The application of chemical additives in poultry litter management aims to control the microbial population and reduce harmful gases. We conducted in vitro analyses to determine and compare the minimum inhibitory concentration (MIC) of commercial chemical additives of calcium oxide, aluminum sulphate, and copper sulphate on bacteria such as Salmonella Enteritidis, Salmonella Infantis, Salmonella Heidelberg, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa; yeast (Candida albicans); and fungi (Aspergillus flavus and Penicillium citrinum). The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were also evaluated. Chemical additive solutions were prepared at a concentration of 0.1 g/mL and diluted up to 0.00078 g/mL, corresponding to concentrations from 10 kg/m² to 0.07 kg/m² when the additives were applied to the litter. In general, aluminum sulphate inhibited the growth of bacterial and fungal strains at a concentration of 0.62 kg/m² (6.25%). On the other hand, additives based on copper sulphate and calcium oxide showed an inhibitory effect on bacterial and fungal strains at dosages above 2.5 kg/m² and 5 kg/m², respectively. Research into commercially available poultry products is rare but important for determining the correct dosage of chemical additives to control the microbial and fungal population of poultry litter. The use of a correct dosage can prevent microbial resistance to chemical additives. This study continues to analyze the use of additives on commercial farms.

1. Introduction

The reuse of poultry litter in successive batches is a practice of economic and environmental interest for poultry production. Producers often face material shortages, and the waste generated by production must be disposed of correctly to avoid contaminating the environment. However, reusing litter can make disinfecting sheds and altering the microbiological quality of the production system difficult [1]. More than thirty different genera of bacteria have been found in poultry litter, mainly Lactobacillus spp. and Salinococcus spp., as well as Clostridium spp., Staphylococcus spp., and Bordetella spp. [2]. Among the bacteria, those of the genus Salmonella are the most important in poultry farming because they pose a risk of food contamination for humans [3]. In Brazil, given the importance of this pathogen, Salmonella control in poultry facilities is regulated by specific legislation that indicates the method and frequency of monitoring on farms [4]. Escherichia coli is a bacterium that is found in the intestines of healthy humans and animals and thus part of the normal bacterial flora. However, some Escherichia coli strains can cause diseases and lead to serious infection [5]. These strains are called STEC/VTEC (shiga toxin- or verotoxin-producing Escherichia coli) or EHEC (enterohaemorrhagic E. coli), and their toxins have the potential to cause bloody diarrhea and hemolytic uremic syndrome (HUS), a serious complication that can be fatal [6].

Staphylococcus aureus is recognized as the third most important cause of foodborne infections in the world [7] and is regarded as an opportunistic and commensal organism in animals, birds, and humans. It may reside asymptomatically on the skin and in the noses of animals [8]. Similarly, Pseudomonas aeruginosa is regarded as a normal inhabitant and opportunistic organism of avian spp. under normal environmental conditions. However, under stressors, this organism becomes pathogenic and induces clinical symptoms. Infection with Pseudomonas aeruginosa in birds is associated with septicemia, respiratory signs, diarrhea, and death [9], with serious economic losses in the poultry industry [10].

The concern regarding fungi is the exposure of birds to mycotoxins. Fungi and yeasts are consistent members of animal microflora that are poorly understood in relation to the production of poultry. Fungi, like bacteria previously, have been associated with the onset of disease. However, evidence shows that fungi can provide protective effects, much like bacterial populations [11]. The presence of fungi in litter is due to the source material and the quality of storage, since in nature, they are found in plant species used as litter material, such as peanut hulls, rice hulls, or coffee hulls.

Attention to the quality of broiler litter has increased in recent times, as it can negatively impact poultry health and the environment. Research indicates that the moisture content of broiler litter is higher than 50%. This high humidity can lead to a faster reproduction of bacteria and pathogens and more ammonia emissions [12]. For this reason, the management and treatment of reused poultry litter has become an essential practice in poultry production. In particular, it is one of the recommended practices within biosecurity measures. Different management practices have been used to reduce moisture levels and pathogens in poultry litter [13]. Among the litter treatment alternatives is the application of chemical additives. Substances such as calcium oxide, calcium hydroxide, agricultural gypsum, Quicklime, aluminum sulphate, and even biological products have been added to litter to reduce enterobacteria [1,3,14,15,16]. A study evaluated the use of an additive that presented better results when applied to poultry litter and concluded that the application of Bacillus subtillis at a dosage of 5 g/m² of litter significantly reduces the number of bacteria in the production of broiler breeders [17]. In general, however, data on the use of commercial additives in Brazil are lacking, since the use of fermentation and lime is widespread in the country.

The objective of this study was to determine and compare the MIC of commercial chemical additives on bacteria (Salmonella Enteritidis, Salmonella Infantis, Salmonella Heidelberg, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa), yeast (Candida albicans), and fungi (Aspergillus flavus and Penicillium citrinum), as well as the MBC of the bacteria and the MFC of the yeast and fungi.

2. Materials and Methods

Three chemical additives were evaluated, including Quicklime (calcium oxide), AvianControl^® (aluminum sulphate, Produtos Quimicos Guaçu, Estiva Gerbi, Brazil), and Stalosan^® F (compound based on copper sulphate and calcium sulphate, San Vet Holding GmbH, Herzogenburg, Austria). The dosages to be applied to poultry facilities for each additive were chosen according to the manufacturer’s instructions or based on dosages currently used in the literature (

Table 1. Dosages applied to poultry facilities according to manufacturer’s instructions or the literature for each evaluated additive.

Commercial Additive	Recommended Dose (kg/m2)	Application Surface	Source
Quicklime	0.60	Litter	Dai-Prá et al., 2009 [3]
AvianControl®	0.50	Litter	Produtos Quimicos Guaçu
Stalosan® F	0.05	Litter, walls, and curtains	San Vet Holding GmbH

).

To facilitate MIC, MBC, and MFC analyses, an aqueous suspension of each additive was prepared at a concentration of 0.1 g/mL in sterile Milli-Q^® water. This pure solution corresponds to 10 kg/m² if the additive was applied to a poultry facility (

Table 2. Microdilution of the solution prepared for the in vitro analyses, number of microplate wells (serial dilutions), and their corresponding concentrations in percentages and when applied to facilities in kg/m².

Microdilution	Microplate Well nº	Tested Concentration (%)	Application to Facilities (kg/m2) 2
1 1	Not applicable	100 1	10
½	1	50	5
¼	2	25	2.5
1/8	3	12.5	1.25
1/16	4	6.25	0.62
1/32	5	3.12	0.31
1/64	6	1.56	0.15
1/128	7	0.78	0.07

¹ Pure solution (0.1 g/mL). ² Quantities applied to the facilities for each dilution/concentration.

).

The broth microdilution method created by the National Committee for Clinical Laboratory Standards (NCCLS) [18] was used to determine the MIC and was carried out in triplicate. To prepare the inoculum, the strains of Salmonella Enteritidis (ATCC 130 76), Salmonella Infantis (isolated strain), Salmonella Heidelberg (isolated strain), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 025923), and Pseudomonas aeruginosa (ATCC 15442) were grown in BHI broth (HiMedia, Mumbai, India). Clostridium perfringens strains (INCQS 00215) were grown in differential broth for clostridia (DRCM) with a selective supplement (Merk Millipore, Darmstadt, Germany) and incubated in an anaerobiosis jar (Permution^®) with a microaerophilic generator (Anaeropack, Tokyo, Japan).

Using U-bottom 96-well microplates, 95 μL of the additive solution was added to the first well, followed by serial dilutions up to the tenth well (Table 2). The amount of inoculum used was 5 μL, with a final concentration of 10⁷ CFU/mL. Negative controls (95 μL of BHI broth and 5 μL of the inoculum) and positive controls (5 μL of inoculum, 94 μL of BHI broth, and 1 μL of the antibiotic antimycotic solution, Sigma A5955) were added to wells 11 and 12, respectively. After dilution, the microplates were incubated at 37 °C for 24 h. The MIC result was observed and confirmed by adding 40 µL of vital dye (I8377, Sigma, Livonia, MI, USA) at a concentration of 2 mg/ mL to each well, followed by an incubation period of 3 h at 37 °C. The MIC value was the lowest concentration of an additive in kg/m² at which bacterial growth was inhibited. Inhibition was considered to have been detected when the wells turned pink. Red wells meant the detection of growth.

The minimum bactericidal concentration (MBC) was also tested via surface plating. In Petri dishes containing BHI agar, 100 µL of the inoculum from the microplate wells where growth was inhibited (pink) and/or possibly absent (translucent) was incubated for 24 h at 37 °C. The MBC of Clostridium perfringens was determined in the same way on plates containing a base agar (Kasvi, Pinhais, Brazil) added to a 50% egg yolk emulsion and a selective supplement, both from Merk^® (Merk Millipore, Darmstadt, Germany). The MBC was defined as the concentration of the additive at which the microorganism did not grow after the incubation period.

The broth microdilution test created by NCCLS [19] was used to determine the MIC for yeast and was performed in triplicate. A yeast inoculum of the Candida albicans species (INCQS 40178) grown in BHI broth was used. Using U-bottom 96-well microplates, 100 μL of the additive solution was added to the first well, followed by serial dilutions up to the tenth well (Table 2). Then, 100 μL of the inoculum, at a final concentration of 10⁵ CFU/mL, was added up to the 9th well. Additionally, negative controls (200 μL of RPMI 1640 broth and 100 μL of the additive) and positive controls (20 μL of the inoculum, 100 μL of RPMI broth, and 100 μL of amphotericin B) were added to wells 10 and 11, respectively. RPMI broth and the inoculum were added to the 12th well for growth control. After microdilution, the plate was homogenized and then incubated at 37 °C for 24 h.

The broth microdilution test created by NCCLS [20] was used to determine the MIC for moulds and was performed in triplicate. For this purpose, Aspergillus flavus (USDA 3251) and Penicillium citrinum (INCQS 40011) were grown on Sabouraud Dextrose Agar (SDA) (KASVI, Pinhais, Brazil) for 5 to 7 days at 32 °C. After that, the colonies were covered with 5 to 10 mL of sterile distilled water supplemented with 0.1% Tween 20. Conidia were carefully scraped off with a Drigalski loop and transferred to a sterile tube. The conidia were counted in a homokinetic chamber, and the final inoculum concentration used in this test was 3 × 10⁵ CFU/mL. Using U-bottom 96-well microplates, 100 μL of the additive solution was added to the first well, followed by serial dilutions up to the tenth well (Table 2). Then, 100 μL of the inoculum was added up to the 9th well. Additionally, negative controls (200 μL of RPMI 1640 broth and 100 μL of the additive) and positive controls (20 μL of the inoculum, 100 μL of RPMI broth, and 100 μL of amphotericin B) were added to wells 10 and 11, respectively. RPMI broth and the inoculum were added to the 12th well for viability control (growth). Finally, the microplates were incubated at 35° for 24–48 h. The MIC was defined as the lowest concentration of the additive capable of inhibiting fungal growth (absence of a growth halo).

The minimum fungicidal concentration (MFC) was also tested for yeast and fungi via surface plating. Ten microliters of the suspension from the microplate wells, where complete inhibition of yeast/fungal growth was observed (presence of a growth halo), was added to Petri dishes containing Sabouraud Dextrose Agar (SDA) (KASVI, Pinhais, Brazil) with chloramphenicol [21]. The Petri dishes were then incubated at 32 °C for 5 to 7 days.

3. Results

3.1. Bacterial Species

In general, the results showed that the additives have a different inhibitory or bactericidal action for each microorganism (

Table 3. MIC and MBC of chemical additives against isolates of bacterial species (kg/m²).

	AvianControl®		Stalosan® F		Quicklime
Species	MIC	MBC	MIC	MBC	MIC	MBC
E. coli	-	≥1.25	≥2.50	≥5.00	≥1.25	≥5.00
S. Enteritidis	≥0.62	≥1.25	≥2.50	≥5.00	-	≥1.25
S. Infantis	-	≥1.25	≥5.00	-	≥2.50	≥5.00
S. Hildelberg	≥0.62	≥1.25	≥2.50	≥5.00	-	≥5.00
S. aureus	≥0.62	≥1.25	≥5.00	-	≥0.62	≥1.25
P. aeruginosa	-	≥0.62	≥5.00	-	≥2.50	-
C. perfringens	-	≥0.31	≥0.31	≥0.62	≥2.50	≥5.00

MIC: minimum inhibitory concentration. MBC: minimum bactericidal concentration. “-”: values not detected.

). Overall, the bactericidal effect of the additives was observed against strains of E. coli, S. Enteritidis, S. Heidelberg, and C. perfringens. The choice of additives for litter treatment must be linked not only to their technical efficiency but also their economic efficiency so as not to burden production costs. When looking at the MIC for the different additives, the intention is that the product proves to be efficient against pathogenic bacteria in a quantity that is worth the investment in its application. When compared against one another, the additive with the best results was AvianControl^®, showing an MBC of 1.25 kg/m² against all the bacterial strains tested. For C. perfringens, AvianControl^® showed an MBC of 0.31 kg/m² and for P. aeruginosa, it was over 0.62 kg/m².

The bactericidal action of Stalosan^® F was 50% (v/v), i.e., 5.00 kg/m², for most of the strains tested. The MIC was 25% (v/v) for E. coli, S. Enteritidis, and S. Heidelberg, while that for C. perfringens showed a very interesting result, with an MIC of 0.31 kg/m².

Quicklime showed MIC values of 0.62 kg/m² for S. aureus. However, to inhibit the growth of the other strains evaluated in this study, the MIC found was 1.25 kg/m².

3.2. Fungal Species

After serial dilutions of the additives, the best result for the control of C. albicans was achieved by AvianControl^® (

Table 4. MIC and MFC of chemical additives against isolates of fungal species (kg/m²).

	AvianControl®		Stalosan® F		Quicklime
Species	MIC	MFC	MIC	MFC	MIC	MFC
Candida albicans	≥0.15	≥0.31	-	≥1.25	-	≥2.50
Aspergillus flavus	≥5.00	-	-	≥5.00	-	≥2.50
Penicillium citrinum	≥5.00	-	≥1.25	≥2.50	-	≥2.50

MIC: minimum inhibitory concentration. MFC: minimum fungicidal concentration. “-”: values not detected.

). AvianControl^® inhibited yeast growth from a concentration of 0.15 kg/m² (MIC) and had a fungicidal effect from 0.31 kg/m² (MFC).

The additive Stalosan^® F also showed a fungicidal effect on C. albicans from a concentration of 1.25 kg/m². Otherwise, no additive concentration inhibited the growth of this species.

Quicklime inhibited yeast growth at 2.50 kg/m², and the MIC was not observed.

The results of the effect of the additives on the filamentous fungus are also shown in Table 4. The MIC observed for both A. flavus and P. citrinum was 5.00 kg/m². In addition, no MIC could be observed for Quicklime and Stalosan^® F for these species. On the other hand, the same additives showed MFCs above 5.00 kg/m² for Stalosan^® F and above 2.50 kg/m² for Quicklime (Figure 1a,b).

For AvianControl^®, no MFC of the additive was observed, and the growth of P. citrinum was inhibited from 50% of the evaluated concentration, i.e., 5 kg/m². For Stalosan^® F, the only MIC observed was against P. citrinum (1.25 kg/m²), and no MIC values could be observed for Quicklime. Finally, the minimum fungicidal concentration shown by Stalosan^® F and Quicklime against P. citrinum was 25% (2.50 kg/m²).

4. Discussion

The use of chemical additives for the management of poultry litter attempts to modify the environment in a way that interferes with the process of microbial proliferation. The release of ions and changes in moisture, water activity, and pH of the litter can explain the antimicrobial effect of an additive. In this sense, the most important aspects are the dosage applied and the duration of the product’s action. The latter aspect is a significant factor in ensuring that the application is sufficient and does not have a negative impact on production costs.

AvianControl^® (Guaçu Group, Brazil), a powder composed mainly of aluminum sulphate, is indicated for use in broiler litter to absorb moisture, lower pH, and reduce ammonia production. Although the physicochemical effects of using aluminum sulphate have been well studied, few studies have evaluated its action on the microbiota of poultry litter [22]. The decrease in pH is due to aluminum sulphate, an acid with six moles of protons formed for each mole of sulphate dissociated [23]; this reaction is represented by the following equation: Al₂(SO₄)₃.14 H₂O + 6H₂O → 2Al(OH)₃ + 3SO²⁻ ₄ + 6H⁺ + 14H₂O.

When aluminum sulphate was added to chicken litter with rice straw to test its effect on pH, it was effective in reducing the pH from 7.47 to 4.43 [24]. A pH below 7 and H⁺ ions in the litter cause the ammonium–ammonia ratio to increase, i.e., more ammonia will be converted into ammonium ions, which are not volatile [16]. The effect of aluminum sulphate on litter microbiota is precisely due to this acidification of the environment. The pH of the medium was not measured in this study. However, aluminum sulphate is considered an acidifier. In this sense, the application of pH-reducing products to poultry litter has been widely adopted because reducing the pH will create an environment that is less favourable to the multiplication of bacteria [25]. However, the mechanism of action of the additive is not fully understood. The change in pH caused by the hydrolysis of aluminum sulphate is proposed to affect the metabolism of cells (bacterial or fungal). Hydrogen ions act on the cytoplasmic membrane of the bacterial cell. As the cytoplasmic membrane is responsible for essential functions such as metabolism and cell division, damage to this structure inhibits or stops bacterial or fungal growth.

Quicklime has the opposite effect to that of aluminum sulphate; its application to poultry litter causes an increase in the pH of the medium, which has a direct effect on the litter microbiota, including Salmonella serovars, as they struggle to survive at pH values above 9.5 [19]. Its antimicrobial activity is due to the release of calcium hydroxide, which causes effects such as protein denaturation, damage to the cytoplasmic membrane, and damage to bacterial DNA [19]. A study evaluating the effect of Quicklime in controlling Salmonella and Clostridium in poultry litter concluded that starting at a concentration of 300 g/m², the additive was effective in reducing the population of these microorganisms [3]. In the present in vitro study, the MIC needed to observe an inhibitory effect of Quicklime on Salmonella serovars and Clostridium perfringens was much higher (2.50 kg/m²), unlike that observed in a field study. In installations, the additives are applied directly (in powder form) to the poultry litter, whereas in in vitro studies, aqueous suspensions need to first be prepared. In general, antimicrobial substances are less active in real systems (for example, when used on food in real environments) than in laboratory environments. Sometimes, their activity is also higher when measured in solutions because the liquid phase offers better opportunities for the antimicrobial to encounter the microorganism than, for example, on a solid surface.

Stalosan^® F (Stormollen, Tureby, Storstrom, Denmark), a powder consisting mainly of phosphate compounds (85%), copper sulphate (2.5%), iron sulphate (2.1%), active chlorine, persica oil (0.05%), and aluminum silicate (10.1%), is indicated for use in livestock farms to reduce the number of microorganisms in the environment, to absorb moisture, and to reduce ammonia production. The additive consists of antimicrobial substances that affect cell metabolism and cause physical damage to bacterial cells. In an in vitro study, the use of Stalosan^® F in swine facilities reduced the viability of Lawsonia intracellularis in the environment [26]. This Gram-negative bacterium is the agent responsible for the infectious disease known as porcine proliferative enteropathy (PPE). The authors also observed that the effectiveness of the additive depended on the dose and exposure time. After exposure to Stalosan^® F powder for 30 min, the bacterial cell wall became more translucent, indicating cell wall damage [26].

The mechanism of antifungal action of the additives evaluated in this research has not been elucidated. Antifungals (drugs) can be grouped into three classes based on their site of action, namely azoles, which inhibit the synthesis of ergosterol (the main fungal sterol); polyenes, which interact with fungal membrane sterols from a physicochemical point of view; and 5-fluorocytosine, which inhibits macromolecular synthesis [27]. The additives evaluated in this study could have interfered with the cell membrane and inhibited or hindered the synthesis of fungal enzymes. As studies aimed at elucidating the mechanism of action of chemical additives used in poultry production have not been found in the literature, this type of study would be very interesting for poultry farming, seeing as biosecurity measures recommended in poultry production could be more effective and improve animal productivity. However, this study has some limitations because the interval between the concentrations used to determine the MIC is linked to dilutions made in a microplate. Further studies could be carried out to test smaller differences between the concentrations of the additives. In addition, whether the preparation of aqueous solutions to evaluate the MIC interferes in any way with the action of the additives remains unknown, since in practice, the product is applied in powder form.

5. Conclusions

As expected, different MICs were observed for each tested additive because each additive has a different composition. Each type of additive has a different mode of action in terms of the bacterial and fungal species studied. To ensure that these additives are effective on commercial farms, as a next step, we intend to continue evaluating the antimicrobial activity of these compounds when they are applied directly to poultry litter. In general, AvianControl^® proved to be more effective against the tested bacteria, yeast, and fungi, as it was associated with lower MIC values than the other additives.