The Identification and Quantification of 21 Antibacterial Substances by LC-MS/MS in Natural and Organic Liquid Fertilizer Samples.

Antibiotics in animal production are widely used around the world for therapeutic and preventive purposes, and in some countries, they still serve as antibiotic growth stimulants. Regardless of the purpose of using antibiotics in livestock, they may be present in animal tissues and organs as well as in body fluids and excretions (feces and urine). Farm animal excrement in unprocessed form (natural fertilizers) or processed form (organic fertilizers) is applied to agricultural fields because it improves soil fertility. Antibiotics present in fertilizers may therefore contaminate the soil, surface, groundwater, and plants, which may pose a threat to the environment, animals, and humans. Therefore, it is important to develop analytical methods that will allow for the control of the presence of antibacterial substances in natural and organic fertilizers. Therefore, in this study, an LC-MS/MS method was developed and validated for the determination of 21 antibacterial substances in natural and organic liquid fertilizers. The developed method was used to analyze 62 samples of natural and organic liquid fertilizers, showing that over 24% of the tested samples were contaminated with antibiotics, mainly from the group of tetracyclines and fluoroquinolones. Studies of post-fermentation sludge from biogas plants have shown that the processes of anaerobic methane fermentation, pH, and temperature changes taking place in bioreactors do not lead to the complete degradation of antibiotics present in the material used for biogas production. For this reason, monitoring studies of natural and organic fertilizers should be undertaken to limit the introduction of antibiotics into the natural environment.

Prevalence of veterinary antibiotics in natural and organic fertilizers from animal food production and assessment of their potential ecological risk.

BACKGROUND: Veterinary antibiotics are emerging contaminants and enter into soil principally by agricultural application of organic fertilizers. This article presents the results of the research obtained for the analyzed 70 samples of fertilizers (pig and poultry manure and slurry and digestate) for various classes of antibiotics. RESULTS: Doxycycline, oxytetracycline, tetracycline, lincomycin, tiamulin and enrofloxacin were found in tested samples. Doxycycline was found as a dominant compound, and its highest concentration was 175 mg/kg in pig manure. This investigation indicated that fertilization with manure, especially animal feces, might be the primary source of antibiotics. Additionally, a risk assessment based on a risk quotient was carried out, which showed that the determined concentrations of antibiotics in fertilizers may pose a threat to soil microorganisms. CONCLUSIONS: Results suggested that the ecological risk effects of antibiotic contamination on soil bases and their potential adverse risk on human health needs special attention. © 2023 Society of Chemical Industry.

Probiotic Potential of Clostridium spp.-Advantages and Doubts.

Clostridium spp. is a large genus of obligate anaerobes and is an extremely heterogeneous group of bacteria that can be classified into 19 clusters. Genetic analyses based on the next-generation sequencing of 16S rRNA genes and metagenome analyses conducted on human feces, mucosal biopsies, and luminal content have shown that the three main groups of strict extremophile anaerobes present in the intestines are Clostridium cluster IV (also known as the Clostridium leptum group), Clostridium cluster XIVa (also known as the Clostridium coccoides group) and Bacteroides. In addition to the mentioned clusters, some C. butyricum strains are also considered beneficial for human health. Moreover, this bacterium has been widely used as a probiotic in Asia (particularly in Japan, Korea, and China). The mentioned commensal Clostridia are involved in the regulation and maintenance of all intestinal functions. In the literature, the development processes of new therapies are described based on commensal Clostridia activity. In addition, some Clostridia are associated with pathogenic processes. Some C. butyricum strains detected in stool samples are involved in botulism cases and have also been implicated in severe diseases such as infant botulism and necrotizing enterocolitis in preterm neonates. The aim of this study is to review reports on the possibility of using Clostridium strains as probiotics, consider their positive impact on human health, and identify the risks associated with the expression of their pathogenic properties.

Botulinum toxin in cancer therapy-current perspectives and limitations.

Different serotypes of botulinum toxins (BoNTs) act upon different types of SNARE proteins. This property is used in aesthetic medicine to treat certain eye disorders such as crossed eyes (strabismus) and uncontrolled blinking (blepharospasm), to treat muscle spasms or movement disorders, and, for the two last decades, more and more often, to provide support in cancer therapy, especially so as to obtain analgesic effects upon spastic conditions. The limited literature data also suggests that the addition of BoNTs to the culture of cancer cell lines reduces cell growth, and mitotic activity, and promotes their apoptosis. BoNTs have several advantages that can be emphasized: BoNTs act on both perfusion and oxygenation; moreover, BoNTs are considered to be safe and free of systemic side effects upon administration. Recently, advances in molecular biology techniques have allowed a wide variety of novel BoNT constructs with alternative functions. These constructs could be assessed as potential new classes of anti-cancer drugs. This creates new potential perspectives in the wider use of non-toxic modified BoNT constructs in cancer therapy. In the light of the mentioned premises and existing literature reports, the aim of this review is to summarize current data and reports considering BoNT use in cancer therapy. KEY POINTS : •Botulinum toxin (BoNTs) may be useful in cancer treatment. •Botulinum toxin can serve as an analgesic after cancer radiotherapy. •Botulinum toxin has the ability to inhibit tumor growth and promote apoptosis of neoplastic cells.

HPLC-FLD-Based Method for the Detection of Sulfonamides in Organic Fertilizers Collected from Poland.

Antibacterial substances such as sulfonamides are widely used in veterinary medicine to treat many bacterial diseases. After their administration to animals, up to 90% of the initial dose of the antibiotic is excreted in the feces and/or urine, which can be applied to farmland as natural or organic fertilizers. In this work, an analytical method was developed with the use of HPLC-FLD for the detection and quantification of five sulfonamides (sulfaguanidine, sulfadiazine, sulfamerazine, sulamethazine and sulfamethoxazol) in poultry and pig feces, slurry and digestates. The method was validated according to EU requirements (Commission Decision 2002/657/EC and VICH GL49). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method for all analyzed matrices, recoveries were satisfactory (77.00-121.16%), with repeatability and reproducibility in the range of 4.36-17.34% to 7.94-18.55%, respectively. Decision limit (CCα) and detection capability (CCß) were 33.87-67.63 and 53.36-92.00 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 13.53-23.30 and 26.02-40.38 µg/kg, respectively, depending on the analyte. The forty-four samples of natural and organic fertilizers were analyzed, and four samples showed sulfamethoxazole in the amount from range 158 to 11,070 µg/kg. The application of antibiotics including sulfonamides for farming animals is widespread and may lead to the development of antibiotic resistance and other environmental effects.

Analytical capabilities of micellar liquid chromatography and application to residue and contaminant analysis: A review.

Green chemistry is the use of chemistry to reduce or eliminate the use of generation of feedstocks, products, by-products, solvents, reagents, etc. that are hazardous to human health or the environment. One of the branches of green chemistry is micellar liquid chromatography. Micellar liquid chromatography is a reversed-phase liquid chromatographic mode with mobile phases containing a surfactant above its critical micellar concentration. The applications of micellar liquid chromatography for the determination of numerous compounds in pharmaceutical formulation, biological samples, food, environmental samples, and feeds have been growing rapidly. Micellar liquid chromatography technique has several advantages over other chromatographic techniques. Its main advantage is the small amount of organic modifiers used such as acetonitrile and methanol and the safety and recyclability of the mobile phase. In our work, we discuss the development of "green chemistry" and present what micellar liquid chromatography is. This article presents application methods with the use of micellar liquid chromatography for analysis on antibacterial substances, melamine, biogenic amines, plant protection products, flavonoids, as well as peptides in biological matrices such as milk, eggs, tissues, honey, and feed.

Application of Micellar Mobile Phase for Quantification of Sulfonamides in Medicated Feeds by HPLC-DAD.

Rapid chromatographic procedure for quantification of five sulfonamides in medicated feeds are proposed. Satisfactory separation of sulfonamides from medicated feeds was achieved using a Zorbax Eclipse XDB C18 column (4.6 × 150 mm, 5 µm particle size) with a micellar mobile phase consisting of 0.05 M sodium dodecyl sulphate, 0.02 M phosphate buffer, and 6% propan-2-ol (pH 3). UV quantitation was set at 260 nm. The proposed procedure allows the determination of sulfaguanidine, sulfadiazine, sulfamerazine, sulfamethazine, and sulfamethoxazole in medicated feeds for pigs and poultry. Application of the proposed method to the analysis of five pharmaceuticals gave recoveries between 72.7% to 94.7% and coefficients of variations for repeatability and reproducibility between 2.9% to 9.8% respectively, in the range of 200 to 2000 mg/kg sulfonamides in feeds. Limit of detection and limit of quantification were 32.7-56.3 and 54.8-98.4 mg/kg, respectively, depending on the analyte. The proposed procedure for the quantification of sulfonamides is simple, rapid, sensitive, free from interferences and suitable for the routine control of feeds. In the world literature, we did not find the described method of quantitative determination of sulfonamides in medicated feeds with the use of micellar liquid chromatography.

Quantification of Veterinary Antibiotics in Pig and Poultry Feces and Liquid Manure as a Non-Invasive Method to Monitor Antibiotic Usage in Livestock by Liquid Chromatography Mass-Spectrometry.

Antibiotics are active substances frequently used to treat and prevent diseases in animal husbandry, especially in swine and poultry farms. The use of manure as a fertilizer may lead to the dispersion of antibiotic residue into the environment and consequently the development of antibiotic-resistant bacteria. Most pharmaceutical active ingredients are excreted after administration, in some cases up to 90% of the consumed dose can be found in the feces and/or urine as parent compound. Therefore, due to antibiotic metabolism their residues can be easily detected in manure. This article describes a method for simultaneous analysis of ciprofloxacin, chlortetracycline, doxycycline, enrofloxacin, lincomycin, oxytetracycline, tetracycline, tiamulin, trimethoprim and tylosin in feces, liquid manure and digestate. Antibiotics were extracted from the different matrices with McIlvaine-Na2EDTA buffer solution and the extract was purified by the use two techniques: d-SPE and SPE (Strata-X-CW cartridges) and final eluent was analyzed by LC-MS and LC-MS/MS. The European Commission Decision 2002/657/EC was followed to conduct the validation of the method. Recoveries obtained from spiked pig and poultry feces and liquid manures samples ranged from 63% to 93% depending on analytes. The analysis of 70 samples (feces, liquid manure and digestate) revealed that 18 samples were positive for the presence of doxycycline, oxytetracycline, tetracycline, chlortetracycline, enrofloxacin, tiamulin and lincomycin. The results obtained in the presented study demonstrated that animal feces can be used as a non-invasive method detection antibiotic usage in animal production.

Contamination of Animal Feed with Undeclared Tetracyclines-Confirmatory Analysis by Liquid Chromatography-Mass Spectrometry after Microbiological Plate Test.

The presence of tetracycline (TC) antibiotics was determined in animal feed that had been previously screened with a microbiological plate test. Feed samples were screened by a microbiological plate test on a pH 6.0 culture medium seeded with Bacillus cereus ATCC 11778 able to pre-reveal the presence of tetracyclines. Subsequently, confirmation and quantification were performed using a validated HPLC method with mass spectrometric detection. In 2013-2018, 353 feed samples were analysed to detect antibacterial substances, of which 186 (52.7%) were suspected to contain tetracyclines. Forty-two out of 186 (22.6%) samples analysed by the chromatographic method contained undeclared tetracyclines, which were determined at concentrations from 0.3 to 49 mg kg-1. The most frequently identified contaminating tetracyclines were doxycycline and chlortetracycline.

Determination of Sulfonamides in Feeds by High-Performance Liquid Chromatography after Fluorescamine Precolumn Derivatization.

A new multi-residue method for the analysis of sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfaguanidine and sulfamethoxazole) in non-target feeds using high-performance liquid chromatography-fluorescence detection (HPLC-FLD) and precolumnderivatization was developed and validated. Sulfonamides (SAs) were extracted from feed with an ethyl acetate/methanol/acetonitrile mixture. Clean-up was performed on a Strata-SCX cartridge. The HPLC separation was performed on a Zorbax Eclipse XDB C18 column with a gradient mobile phase system of acetic acid, methanol, and acetonitrile. The method was validated according to EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method, recoveries were satisfactory (79.3⁻114.0%), with repeatability and reproducibility in the range of 2.7⁻9.1% to 5.9⁻14.9%, respectively. Decision limit (CCα) and detection capability (CCß) were 197.7⁻274.6 and 263.2⁻337.9 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 34.5⁻79.5 and 41.3⁻89.9 µg/kg, respectively, depending on the analyte. Results showed that this analytical procedure is simple, rapid, sensitive, and suitable for the routine control of feeds.

Simultaneous determination of sulfonamides, trimethoprim, amoxicillin and tylosin in medicated feed by high performance liquid chromatography with mass spectrometry.

Introduction: The article presents a rapid and simple analytical procedure for determination of four sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine and sulfamethoxazole), trimethoprim, tylosin and amoxicillin in animal medicated feed. Material and Methods: Eighteen medicated feed samples were analysed for active substances. The analytical protocol used a mixture of acetonitrile and 0.05 M phosphoric buffer, pH 4.5 for the extraction of seven antibacterial substances. After extraction, the samples were diluted in Milli-Q water and analysed by liquid chromatography with mass spectrometry. The developed procedure was subjected to validation in terms of linearity, selectivity, limits of quantification and determination, repeatability, reproducibility and uncertainty. Results: The validation of the method was carried out in accordance with the criteria set out in Commission Implementing Regulation (EU) 2021/808 and ICH guidelines. This method provided average recoveries of 90.8 to 104.5% with coefficients of variation for repeatability and reproducibility in the ranges of 3.2-6.9% and 5.2-8.3%, respectively for all analysed antibacterial substances. The limit of detection and limit of quantification for all seven analytes ranged from 5.4 mg/kg to 48.3 mg/kg and from 10.4 mg/kg to 119.3 mg/kg, respectively. The uncertainty of the method depending on the compound varied from 14.0% to 24.0%. The validated method was successfully applied to the 18 medicated feeds. Conclusion: The developed method can be successfully used to routinely control the content and homogeneity of seven antibacterial substances in medicated feed.

Residues of veterinary antibiotics in solid natural and organic fertilizers-method development and sample analysis.

Livestock excrement is used around the world as natural fertilizers or, after processing, as organic fertilizers for crops and grasslands. But due to the presence of veterinary antibiotics in them, they may pose a threat not only to the natural environment, mainly to soil microorganisms, but also to human and animal health. This article describes a method for detecting 21 antibacterial substances in solid natural and organic fertilizers. Antibiotics from fertilizers were extracted with a mixture of acetonitrile and McIlvain-Na2EDTA buffer, twice. The extracts were purified by solid phase extraction technique on Strata-X cartridges and analyzed with the use UHPLC-MS/MS technique. The method was validated in accordance with EU Commission Implementing Regulation 2021/808; the obtained recovery ranged from 93.6 to 116.6% (depending on the analytes), and the linearity ranged from 50 to 1000 µg/kg. The developed method was used to analyze 73 samples of solid natural and organic fertilizers. Our research has shown that over 38% of natural fertilizers were contaminated with antibiotics, mainly doxycycline in concentrations reaching several dozen milligrams per kilogram of fertilizers. In the case of processed organic fertilizers, the presence of antibiotics was found in over 37% of the analyzed samples. The research results showed that the developed and validated analytical method may be useful for assessing the presence and content of antibacterial substances in solid natural and organic fertilizers.

Development and validation method for the determination of selected tetracyclines in animal medicated feedingstuffs with the use of micellar liquid chromatography.

A chromatographic procedure for the determination of oxytetracycline (OTC), tetracycline (TC), chlorotetracycline (CTC), and doxycycline (DC) in medicated feedingstuffs was developed. Samples were extracted with 0.01 M citric buffer/acetonitrile (pH 3.0) and further purified with 0.45 µm syringe filters. The purified extract was separated on Thermo column C18, 150 × 4 mm, 5 µm and detection was carried out at 360 nm for OTC, and TC, 370 nm for CTC, and 350 nm for DC. TCs were eluted with a mobile phase of 0.03 M SDS/7% 1-butanol/0.02 M oxalic acid/NaOH at pH 2.5. This method provided average recoveries of 80.4% to 100.2%, with CVs of 0.5% to 6.6% in the range of 50 to 1500 mg/kg OTC, TC, CTC, and DC in feeds. The linearity for the four TCs was determined by high-performance liquid chromatography-diode array detector (HPLC-DAD) in the range 10-300 µg/mL (50-1500 mg/kg), with a linear correlation coefficient (R) > 0.99. The LOD and LOQ for TCs in pig and poultry feeds ranged from 4.0 to 10.7 and 4.7 to 12.6 mg/kg, respectively. The methodology was applied to the analysis of animal feedingstuffs collected from poultry and pig farms.

Insect Meals and Insect Antimicrobial Peptides as an Alternative for Antibiotics and Growth Promoters in Livestock Production.

The extensive use of antibiotics in animal production has led to the development of antibiotic-resistant microorganisms and the search for alternative antimicrobial agents in animal production. One such compound may be antimicrobial peptides (AMPs), which are characterized by, among others, a wide range of biocidal activity. According to scientific data, insects produce the largest number of antimicrobial peptides, and the changing EU legislation has allowed processed animal protein derived from insects to be used in feed for farm animals, which, in addition to a protein supplement, may prove to be an alternative to antibiotics and antibiotic growth promoters due to their documented beneficial impact on livestock health. In animals that were fed feeds with the addition of insect meals, changes in their intestinal microbiota, strengthened immunity, and increased antibacterial activity were confirmed to be positive effects obtained thanks to the insect diet. This paper reviews the literature on sources of antibacterial peptides and the mechanism of action of these compounds, with particular emphasis on insect antibacterial peptides and their potential impact on animal health, and legal regulations related to the use of insect meals in animal nutrition.

Development, in-house validation and application of a method using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the quantification of 12 ergot alkaloids in compound feeds.

Introduction: Ergot alkaloids (EAs) are toxic substances naturally produced by Claviceps fungi. These fungi infest a wide range of cereals and grasses. When domestic animals are exposed to EAs through contaminated feeds, it is detrimental to them and leads to significant economic losses. For that reason, it is important to monitor feed for the presence of EAs, especially with methods enabling their determination in processed materials. Material and Methods: Ergot alkaloids were extracted with acetonitrile, and dispersive solid phase extraction (d-SPE) was used for clean-up of the extracts. After evaporation, the extracts were reconstituted in ammonium carbonate and acetonitrile and subjected to instrumental analysis using high-performance liquid chromatography with fluorescence detection. The developed method was validated in terms of linearity, selectivity, repeatability, reproducibility, robustness, matrix effect, limits of quantification and detection and uncertainty. The EA content of 40 compound feeds was determined. Results: All the assessed validation parameters fulfilled the requirements of Regulation (EU) 2021/808. At least one of the monitored alkaloids was determined in 40% of the samples. The EAs with the highest incidence rate were ergocryptine, ergometrinine and ergocornine. The total concentrations of EAs ranged from under the limit of quantification to 62.3 µg kg-1. Conclusion: The results demonstrated that the developed method was suitable for simultaneously determining twelve EAs in compound feed and could be used for routine analysis.

Clostridium perfringens-Opportunistic Foodborne Pathogen, Its Diversity and Epidemiological Significance.

The C. perfringens species is associated with various environments, such as soils, sewage, and food. However, it is also a component of the gastrointestinal (GI) microflora (i.e., microbiota) of sick and healthy humans and animals. C. perfringens is linked with different systemic and enteric diseases in livestock and humans, such as gas gangrene, food poisoning, non-foodborne diarrhoea, and enterocolitis. The strains of this opportunistic pathogen are known to secrete over 20 identified toxins that are considered its principal virulence factors. C. perfringens belongs to the anaerobic bacteria community but can also survive in the presence of oxygen. The short time between generations, the multi-production capability of toxins and heat-resistant spores, the location of many virulence genes on mobile genetic elements, and the inhabitance of this opportunistic pathogen in different ecological niches make C. perfringens a very important microorganism for public health protection. The epidemiological evidence for the association of these strains with C. perfringens-meditated food poisoning and some cases of non-foodborne diseases is very clear and well-documented. However, the genetic diversity and physiology of C. perfringens should still be studied in order to confirm the importance of suspected novel virulence traits. A very significant problem is the growing antibiotic resistance of C. perfringens strains. The aim of this review is to show the current basic information about the toxins, epidemiology, and genetic and molecular diversity of this opportunistic pathogen.

Simultaneous Determination of Pyrrolizidine and Tropane Alkaloids in Honey by Liquid Chromatography-mass Spectrometry.

Introduction: Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural contaminants of honey and respectively hepatoxic and neurotoxic compounds. Because honey is a popular constituent of the human diet, it is relevant to warrant the safety of the product. For that reason, a method for simultaneous determination of PAs and TAs in honey based on liquid chromatography- mass spectrometry was developed. Material and Methods: The analytical protocol used sulphuric acid extraction and solid-phase extraction purification. The developed procedure was subjected to validation in terms of linearity, selectivity, repeatability, reproducibility, limits of quantification and determination, matrix effect and uncertainty. A total of 29 honey samples were analysed for the determination of PAs and TAs. Results: All the evaluated validation parameters fulfilled the requirements of European Commission Decision 2002/657/EC. At least one of the monitored alkaloids was determined in 52% of the samples. Among the most abundant alkaloids were echimidine, intermedine and lycopsamine. The total PA concentrations ranged from 2.2 to 147.0 µg kg-1. Contrastingly, none of the monitored TAs was detected in the analysed samples. An assessment of the dietary exposure to PAs from the consumption of the contaminated honeys showed that three of them would pose a risk to consumers, especially if they were children. Conclusion: A sensitive method suitable for simultaneous determination of PAs and TAs in honey was developed and validated. The analysis of 29 honey samples for PAs and TAs revealed that honey destined for retail could pose a risk to consumers.

Laboratory Experience with the Microscopic Method for the Detection of Insects in Poultry Feeds.

Introduction: The use of insects and their processed animal proteins (PAPs) for animal nutrition creates the need for research into methods useful for routine surveillance for their presence. The aim of this study was to evaluate a modified microscopic method for the detection of particles of insects in poultry feed. Material and Methods: A total of 90 samples including PAP of insects (Hermetia illucens and Tenebrio molitor), poultry feeds produced with different levels (0-27%) of insect PAP content, and other poultry feeds spiked with insect PAP at 1% were investigated using a modified microscopic method with a double sedimentation protocol. Results: Characteristic features of insects including cuticulae, muscles, bristles and tracheoles were determined in the microscopic images obtained. In all spiked samples, characteristic fragments of insects were detected. The fragments of muscle and tracheoles only indicated the presence of material from members of the insect class but could not facilitate identification of organisms to species level. Conclusion: The results obtained with this double sedimentation protocol for the isolation of insect PAP from feed for poultry have shown that the method can be used in routine analysis.

Scopolamine and atropine in feeds - determination with liquid chromatography mass spectrometry.

Tropane alkaloids (TAs) are naturally occurring plant toxins. Due to the fact that TA-producing plants can enter the food chain, they pose a risk for animals and human health. Therefore, sensitive analytical methods need to be developed to provide an adequate safety of feed and food. The presented method is based on liquid chromatography-mass spectrometry detection and enables the determination of scopolamine and atropine in compound feeds at a low level of contamination. Limits of quantification for scopolamine and atropine were 0.92 and 0.93 µg kg-1, respectively. Scopolamine-D3 and atropine-D3 were used for quantification. The method was successfully validated and applied to the analysis of 42 feed samples. Among investigated feeds, 67% contained at least one of the monitored alkaloids. Soybean meals were the feed materials contaminated most often, also with the highest determined concentrations of TAs, which reached 147.9 µg kg-1.

Enterococci-Involvement in Pathogenesis and Therapeutic Potential in Cancer Treatment: A Mini-Review.

Enterococcus spp. are Gram-positive, heterogeneous lactic acid bacteria inhabiting various environments. Several species of Enterococci are considered to be able to stimulate the immune system and play an important role in intestinal homeostasis. Some Enterococci can be used as probiotics. Some strains of E. faecium are components of pharmaceutical products used to treat diarrhea, antibiotic-associated diarrhea, or irritable bowel syndrome (IBS). However, it has been proved that they are responsible for food contamination, and are sometimes undesirable from the point of view of food technology. Additionally, the virulence and multi-drug resistance of Enterococci potentially pose a risk of an epidemic, especially in hospital environments. Moreover, there are indications of their negative role in colon tumorigenesis; however, some nterococci are proved to support immunotherapy in cancer treatment. In general, it can be concluded that this group of microorganisms, despite its nature, has properties that can be used to support cancer treatment-both aggressive chemotherapy and cutting-edge therapy targeting immune checkpoints (IC).