In Vitro Activity of Allium cepa Organosulfur Derivatives against Canine Multidrug-Resistant Strains of Staphylococcus spp. and Enterobacteriaceae

Simple Summary The rise of drug-resistant bacteria, particularly in animals, poses a major challenge in veterinary medicine. Antibiotic development lags behind the increasing resistance. To tackle this, alternative therapies have been explored, such as the use of natural products and plant extracts. This study evaluates the laboratory efficacy of plant derivatives of the Alliaceae group (which includes garlic and onion) as antimicrobial agents, with encouraging results. Although further research is needed, these findings suggest a potential role for these natural compounds in veterinary medicine. Abstract Background: The increase of multi-resistant bacteria, especially Staphylococcus spp. and Enterobacteriaceae, constitutes a challenge in veterinary medicine. The rapid growth of resistance is outpacing antibiotic discovery. Innovative strategies are needed, including the use of natural products like Allium species (Allium sativum L. and Allium cepa L.), which have been used empirically for centuries to treat infectious diseases in humans and farm and aquaculture animals due to their antibacterial properties. Methods: This study aimed to evaluate the in vitro activity of two Allium-derived compounds, propyl propane thiosulfinate (PTS) and propyl propane thiosulfonate (PTSO), against multi-resistant Staphylococcus spp. (n = 30) and Enterobacteriaceae (n = 26) isolated from dogs referred to a veterinary teaching hospital in Madrid. Results and Discussion: The results indicated the in vitro efficacy of PTSO/PTS against the tested bacterial strains, and 56.7% of Staphylococcus pseudintermedius and 53.8% of Enterobacteriaceae showed sensitivity to PTS and PTSO compared with classic antibiotics. In addition, 50% of S. pseudintermedius strains resistant to erythromycin, ibofloxacin, difloxacin and orbifloxacin and 50% of Enterobacteriaceae strains resistant to tetracycline and doxycycline were sensitive to PTS and PTSO. Although studies are needed to verify their efficacy in vivo, the combined use of PTS and PTSO exhibits promise in enhancing bacterial sensitivity against S. pseudintermedius and Enterobacteriaceae infections, providing a first insight into the potential of both compounds in veterinary practice.


Introduction
The World Health Organization (WHO) recognizes antimicrobial resistance as one of the most important issues affecting public, animal and environmental health [1].The proximity between dogs and humans entails a potential risk of pathogen transmission, including antibiotic-resistant bacteria [2].Canines can transmit many pathogens to humans, causing infections ranging from skin rashes to life-threatening bacteremia [3].
Antibiotics are critical for combating infectious diseases [4].However, their excessive and inappropriate use combined with inadequate waste management and spread to the environment has contributed to the development of antibiotic-resistant strains and increased mortality due to infectious diseases [5].Recent studies have shown that Gramnegative bacteria often exhibit multidrug resistance (MDR), including to critically important antimicrobials (CIAs), highlighting the complex challenge they pose in antimicrobial resistance [6,7].Primary Gram-negative bacteria with zoonotic potential that cause healthcare complications, such as nosocomial, urinary tract and bloodstream infections, belong to the genus Enterobacteriaceae, which includes Escherichia coli, Klebsiella spp.and Enterobacter spp.Among Gram-positive bacteria, methicillin-resistant Staphylococcus spp.(MRS) and vancomycin-resistant Enterococcus spp.(VRE) are of particular concern [8,9].
Resistance outpaces the rate at which new antibiotics are discovered, making antibiotics a finite healthcare resource [10].As a result, we are currently faced with a situation where the therapeutic options for infections are limited because certain pathogenic strains are resistant to all existing groups of antibiotics [11].Addressing this challenge requires urgently exploring new antimicrobials, developing additional agents and investigating innovative chemical structures for enhanced efficacy.These efforts are crucial not only for curing existing infections but also for reducing the risk of future infections in both animals and humans.An integral part of this approach includes controlling bacterial growth in animal feed, which is a key factor in preventing zoonosis and may involve the use of specialized feed additives [12][13][14].
Medicinal plants are rich sources of novel compounds with potential antimicrobial properties [15].However, few veterinary medicine studies have evaluated the efficacy of these plant extracts against antibiotic-resistant bacteria [16,17].Among different medicinal plants, Allium species have been used worldwide for centuries to treat infectious diseases.A substantial amount of historical evidence is available, including documents dating back more than 3500 years from ancient Egypt describing the medicinal properties of this genus [18][19][20].In recent years, the antibacterial properties of Allium plant extracts have been extensively studied, including their efficacy against multidrug-resistant bacteria [21,22].
One of the most well-known components of this botanical family is diallyl thiosulfinate (allicin), which is present in Allium sativum L., and its antimicrobial activity has been widely reported [23][24][25][26].However, its instability limits its suitability as a therapeutic agent in pet food.Allicin degrades easily to other organosulfur compounds such as ajoenes, vinyl dithiins and diallyl polysulfides (DAPS) at 20 • C [27][28][29][30].The proportion of these degradation products can vary depending on the Allium sativum L. processing conditions.In addition, allicin degradation in DAPS raises safety concerns because high concentrations of these molecules with multiple sulfur atoms have a higher potential to oxidize canine erythrocytes than non-degraded thiosulfonates [30].This could explain the conflicting results regarding the safety of Allium sativum L. in pet diets, with a few studies considering it harmful [31].However, others report improved animal health with dietary supplementation [32,33].
In contrast, the organosulfur compounds present in Allium cepa L., propyl propane thiosulfinate (PTS) and propyl propane thiosulfonate (PTSO), have been reported to be safe and not cause toxicity [34][35][36].These molecules exhibit greater stability because, although PTS leads to propyl disulfide (PDS) and PTSO through a disproportion reaction, PDS can be transformed into PTSO in the presence of oxygen [37].PTS and PTSO have demonstrated significant antimicrobial activity in vitro and in vivo against different pathogenic strains in livestock, including Staphylococcus spp., Enterobacteriaceae spp.and Enterococcus spp.[38][39][40].In addition, PTS and PTSO have shown in vitro antimicrobial activity against multidrug-resistant bacteria and yeasts isolated from human clinical samples [41].However, information on the antimicrobial activity of these compounds against antibiotic-resistant bacteria, which commonly affect dogs, is lacking.Methicillin-resistant S. pseudintermedius (MRSP) is an important pathogen commonly encountered in canine infections.Due to its genetic similarities to S. aureus, MRSP poses a considerable concern for veterinary medicine and public health, as it can be transmitted between animals and humans.Enterobacteriaceae strains such as K. pneumoniae and E. coli are among the most common bacterial pathogens observed in dogs [42].
This study evaluated the in vitro antimicrobial activity of the Allium-derived compounds PTS and PTSO, compared to commonly used antibiotics, against different strains of multi-resistant Staphylococcus spp.and Enterobacteriaceae isolated from dogs.

Study Design and Setting
In this study, a total of 56 multi-resistant bacterial strains obtained as etiological agents from hospitalized dogs in a previous study performed at Alfonso X El Sabio University in Madrid were selected [2].Of these, 30 strains, identified as S. pseudintermedius, showed the presence of the mecA gene, which confers resistance by producing a penicillin-binding protein PBP 2A, and 26 species of Enterobacteriaceae [43] (19 E. coli and 7 K. pneumoniae) had at least one extended-spectrum beta-lactamase resistance gene (bla CTX-M1 , bla CTX-M9 , bla SHV, bla TEM ).E. coli CECT 516 and S. pseudintermedius DSM 21284 were used as reference strains.Samples were stored at −20 • C at Alfonso X El Sabio Veterinary Hospital (Madrid, Spain).
PTS and PTSO from Allium cepa L. were supplied with high purity (97%) by Panadog-Enzim-Orbita (Tavira, Portugal) and dissolved in polysorbate-80 to a final concentration of 500 g/L.
To evaluate the antibiotic sensitivity of Allium compounds, the Minimum Inhibitory Concentration (MIC) was selected according to CLSI guidelines [44].For this purpose, decreasing concentrations of the antimicrobial agents (2000-0.48µg/mL) were prepared in 1:2 dilutions in wells of microtiter plates in Mueller-Hinton medium buffer (Scharlab, Barcelona, Spain) with a bacterial inoculum of the different strains in the logarithmic growth phase (approx.10 5 CFU/mL).In addition, uninoculated broth was used as a negative control, and another well with only a bacterial suspension (without antibiotics) was used as a positive control.All samples were performed in duplicate.Samples were incubated in a tube rotator (VWR; Barcelona, Spain) for 24 h at 20 rpm.Subsequently, they were measured using a Multiskan FC Microplate Reader Spectrophotometer (Thermo Scientific, Waltham, MA, USA) at a wavelength of 620 nm.The microorganism was expected to grow in the control tube and in tubes that did not contain sufficient antimicrobials to inhibit its development.Subsequently, according to the methodology described by Turnidge and Paterson (2007) [45], the probability of clinical success for each compound was predicted by establishing a cut-off point of 62.5 µg/mL.Consequently, MICs above this value were considered resistant, whereas those equal to or lower than this value were considered sensitive.

Statistical Analysis
Data analysis was performed using IBM SPSS Statistics v19 software.The percentage of observed bacterial resistance was described along with the frequency distribution.McNemar's test was performed to evaluate the association between resistance to Alliaceae compounds and the different antimicrobials tested.Statistical significance was set at p < 0.05.

Staphylococcus pseudintermedius
The number and percentages of resistant strains obtained from the 30 previously selected antibiotic-resistant Staphylococcus pseudintermedius strains tested with PTS and PTSO, as well as with various antibiotics, are detailed in Table 1.The reference strain S. pseudintermedius DSM 21284 had an MIC value of 31.25 µg/mL for PTS/PTSO, which provides a baseline of the activity of these Allium cepa-derived compounds.The highest resistance rates were observed against erythromycin (90.0%), ibafloxacin, difloxacin, enrofloxacin and orbifloxacin (83.3% each).In addition, among all multi-resistant strains, 43.3% of the evaluated S. pseudintermedius showed resistance to both PTS and PTSO.Despite observed variations in MIC values for PTS and PTSO, with two of the studied strains showing higher values for PTSO compared to PTS, the sensitivity results are still comparable for both compounds when considering the defined cut-off points.
The sensitivity to Alliaceae compounds and various antimicrobials was compared, revealing that a larger proportion of strains were sensitive to PTS and PTSO, as determined by the MIC cut-off of 62.5 µg/mL, in comparison to other tested antimicrobials.In contrast, resistance was observed to the following antibiotics: erythromycin, clindamycin, lincomycin, ibafloxacin, difloxacin, enrofloxacin, marbofloxacin, orbifloxacin, sulfamethoxazole-trimethoprim, streptomycin-trimethoprim and spiramycin-trimethoprim.All Staphylococcus strains containing the mecA gene were considered resistant to amoxicillin, ampicillin, cephalexin, cefotixin, cefquinome and cefoperazone.No nitrofurantoin-resistant strains were detected in this study.In addition, there was a higher proportion of tested strains that showed sensitivity to both Allium-derived compounds and resistance to erythromycin, clindamycin, ibafloxacin, difloxacin, enrofloxacin, marbofloxacin, orbifloxacin, chloramphenicol and trimethoprim/sulfamethoxazole, showing the highest percentage (50%) of the S. pseudintermedius strains resistant to erythromycin, ibafloxacin, enrofloxacin and orbifloxacin (Table 2; Supplementary Material: Tables S1 and S2).[45].A p-value < 0.05 indicates statistically significant differences between sensitivity to Alliaceae compounds and sensitivity/resistance to other antimicrobials.

Enterobacteriaceae
Enterobacteriaceae strains in the study exhibited different percentages of resistance to antibiotics, PTS and PTSO.The reference strain E. coli CECT 516 showed an MIC for PTS/PTSO of 62.5 µg/mL, which establishes a comparative baseline for the activity of these Allium cepa-derived compounds against Enterobacteriaceae.The highest resistance percentages were observed for tetracycline and doxycycline (96.2%), followed by ibafloxacin and difloxacin (88.5% each) and enrofloxacin, marbofloxacin and orbifloxacin (84.6% each), as shown in Table 3.Additionally, 46.2% of the Enterobacteriaceae strains were resistant to PTS and PTSO.A statistically significant difference was observed in the sensitivity of PTS and PTSO compared with that of ibafloxacin, difloxacin, enrofloxacin, marbofloxacin, orbifloxacin, tetracycline and doxycycline.Due to intrinsic extended-spectrum beta-lactamase resistance genes, these strains have been classified as resistant to amoxicillin, ampicillin, cephalexin, cefotixin, cefquinome and cefoperazone.
Finally, Enterobacteriaceae strains demonstrated significantly lower resistance to kanamycin and neomycin than to PTS and PTSO.A higher proportion of the tested strains showed sensitivity to PTS/PTSO and resistance to neomycin, kanamycin, ibafloxacin, difloxacin, enrofloxacin, marbofloxacin, orbifloxacin, tetracycline and doxycycline, with the highest percentage (50%) of Enterobacteriaceae strains resistant to tetracycline and doxycycline.The results are detailed in Table 4.
Finally, when comparing the higher percentages (50%) of strains resistant to antibiotics and sensitive to PTS/PTSO (Tables 2 and 4), a higher proportion of antibiotic-resistant S. pseudintermedius strains was observed than that for Enterobacteriaceae, indicating greater sensitivity to PTS/PTSO.[45].A p-value < 0.05 indicates statistically significant differences between sensitivity to Alliaceae compounds and sensitivity/resistance to other antimicrobials.

Discussion
Antibiotic overuse has led to the development of multidrug-resistant bacterial strains, making it increasingly challenging to treat infections in humans and animals [46,47].Therefore, it is important to explore alternative therapeutic options.Natural products have emerged as valuable novel antimicrobial agent sources due to their diverse chemical com-positions and potential therapeutic properties [48,49].Allium spp., including Allium cepa, have been extensively studied for their medicinal properties, including antimicrobial activity [27,50,51].Organosulfur compounds observed in Allium cepa L. and Allium sativum L. have shown promising antibacterial effects, making them potential candidates for use against multidrug-resistant bacteria [52,53].
Despite the considerable attention natural products have received in human medicine, their application in veterinary medicine remains largely unexplored.Limited studies have assessed the efficacy of natural products against canine bacterial strains.This lack of research highlights the need for further studies investigating the potential therapeutic uses of natural compounds in veterinary applications.
In our study, the combination of conventional antimicrobials with PTS and PTSO has demonstrated promising results in increasing the sensitivity of bacterial strains against infections caused by S. pseudintermedius and Enterobacteriaceae.This approach, as highlighted in our findings, suggests a significant alternative to traditional synergy, aligning with the guidelines for antimicrobial therapy combination [54] and underscoring the need for innovative strategies in the face of rising antibiotic resistance.The in vitro antibacterial activity of PTS and PTSO, as compared with other antimicrobials, has been shown to be effective against multidrug-resistant bacteria [41], supporting the potential of this combination.Furthermore, the importance of systematic mapping of the long-term clearance efficacy of drug combinations, as discussed in recent research [55], is crucial for designing more effective multidrug regimes, especially against persistent infections.
Canine-originating bacterial strains, such as multidrug-resistant Staphylococcus spp.and Enterobacteriaceae, are of great concern to both veterinary and public health [56,57].These strains exhibit substantial diversity and can cause various infections in dogs and other domestic animals, with potential zoonotic implications for humans [58][59][60][61].Furthermore, the ability of these strains to develop multi-drug resistance impairs treatment.Therefore, research on canine-specific bacterial strains is crucial for developing effective therapeutic strategies [62].
Several veterinary medicine studies have investigated the antimicrobial activities of natural products against canine Staphylococcus and Enterobacteriaceae spp.These studies explored a variety of natural sources, including herbal extracts, honey products and bacteriophages, among others [63][64][65].These investigations provided valuable insights into the potential efficacy of natural compounds as alternative therapies, with botanical products being the most promising solutions, such as Garcinia mangostana or Harungana madagascariensis extracts [66,67].Organosulfur derivatives from garlic, such as allicin-inspired compounds, have been reported to exhibit antibacterial activities against Staphylococcus spp.[68].Similarly, in previous studies, PTS and PTSO from onions showed antimicrobial activity against Staphylococcus and Enterobacteriaceae multidrug-resistant strains isolated from human samples [41].However, to our knowledge, this is the first study to evaluate the in vitro activity of these compounds against multidrug-resistant canine strains.
Based on the results of this study, it can be hypothesized that PTS/PTSO can effectively treat a higher percentage of methicillin-resistant S. pseudintermedius infections than betalactams.Furthermore, the tested strains showed lower resistance to PTS and PTSO than to most quinolones, which should be prescribed as a last resort.Additionally, as previously reported, MRS acquires resistance to fluroquinolones [81].Most publications show similar results regarding resistance to these antibiotics and consider these drugs not to be good therapeutic alternatives for MRS treatment [71,82].
This study observed that PTS and PTSO did not show lower resistance rates than aminoglycosides, tetracyclines, rifampicin, chloramphenicol or fusidic acid.Regarding the susceptibility of S. pseudintermedius strains to the different antibiotics tested, our results agree with those obtained by other authors [83].Although moderate-to-high resistance patterns have been described for aminoglycosides, their clinical efficacy as single agents against infections caused by Staphylococcus strains is not well established [84][85][86].Rifampicin, an antibiotic developed in the past, has been of recent interest due to its activity against MRS.The moderate resistance described in this study is consistent with other publications [69,[87][88][89][90][91], although resistant strains have been described [92].The tetracycline resistance rates observed in this study are consistent with those reported in other studies [90].Tetracyclines are more effective in vitro than in vivo against different species of Staphylococcus [88].Fusidic acid resistance in this study was higher than that reported in other publications [65,93].However, more studies are required to determine the correlation between in vitro studies and clinical efficacy.Chloramphenicol was used decades ago; however, it is not now widely used because of its narrow safety margin, the need for frequent administration and the lack of presentations suitable for small animals in most countries [88].Nitrofurantoin did not induce in vitro resistance against any of the Staphylococcus isolates tested in this study.High susceptibility to this drug has been reported in other studies [94].However, this drug is highly toxic and not used in clinical practice [95].
The global problem of antimicrobial resistance includes the emergence of extendedspectrum beta-lactamase (ESBL)-producing Enterobacteriaceae that cause considerable morbidity and mortality, especially E. coli and K. pneumoniae species [96].The prevalence of canine isolates resistant to beta-lactams, including broad-spectrum cephalosporins, has increased in recent years [97,98], leading to urinary infections.Companion animals that live in close contact with humans may contribute substantially to their owners' exposure to ESBL-producing Enterobacteriaceae [98].
In this study, all ESBL-producing Enterobacteriaceae isolates were found to be multidrugresistant.The resistance percentages of ESBL-producing strains to PTS and PTSO were similar to those observed for amoxicillin-clavulanate, with a low resistance to amoxicillinclavulanic acid, consistent with recent studies [99].However, other authors reported higher values [100].Bacteria with ESBL genes are associated with resistance to other non-betalactam antimicrobials, such as tetracyclines, quinolones, lincosamides, macrolides and, to a lesser extent, chloramphenicol and aminoglycosides [98,[101][102][103][104][105][106].Moreover, PTS and PTSO showed higher sensitivity percentages for ESBL strains than most quinolones and tetracyclines.The sensitivity percentages for these groups of antibiotics were similar to those observed in other studies [101,[104][105][106] and do not usually represent a good therapeutic alternative.As observed in other surveys, aminoglycosides presented low resistance percentages, probably because of their limited use in the clinic due to their pharmacokinetics and potential side effects [107,108].Allium compounds showed similar resistance to colistin, nitrofurantoin, chloramphenicol and potentiated sulfonamides in the ESBL strains.Colistin was discontinued because of nephrotoxicity.However, the emergence of carbapenem resistance in clinically important bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, K. pneumoniae and E. coli has propitiated its reintroduction into clinical practice as a last-resort treatment option [109].Resistance to nitrofurantoin has been described in low percentages of ESBL-producing Enterobacteriaceae [95]; however, its toxicity and poor pharmacokinetic characteristics have led to its low use in clinical practice.Chloramphenicol and potentiated sulfonamides have shown low to moderate resistance rates in recent publications [100], probably due to lower antibiotic pressure.
Finally, considering the historically safe use of PTS and PTSO in farm animal species and their dietary origin, both compounds are presumed safe for dog health.The toxicological aspects of PTS and PTSO have previously been tested in experimental animals.In vivo studies have demonstrated the low acute and subchronic oral toxicity of PTSO [34][35][36].In addition, recent studies on rats orally administered with PTS and PTSO for 90 days showed no toxic effects [35].Some studies found in the scientific literature show contradictory results about the safety of alliaceous derivatives in the canine diet, even considering them to be harmful food [31].This fact largely contradicts the results of trials conducted by other authors in which the incorporation of alliaceous in the diet of dogs leads to an improvement in animal health [32,110].For example, the trial conducted by Yamato et al. [33] involved orally administering a daily dose of garlic extract at 90 mg/kg of weight to Beagle dogs for 12 weeks, which not only resulted in the absence of adverse effects, but also improved the health of the animals by increasing the gene expression of antioxidant enzymes.
While PTS/PTSO is currently being commercialized for use in farm animals, such as pigs and poultry, research into its application in pets is a more recent development.This paper serves as a pioneering contribution in this field, exploring the use of PTS/PTSO in pet nutrition, and specifically for dogs.This study aims to provide foundational knowledge and encourage further research.Our findings contribute to an increase in the knowledge of this area, highlighting the need for continued research into the safe and effective use of PTS/PTSO in canine diets.Despite these promising results, further pharmacokinetic studies must establish their safe use in clinical practice.

Conclusions
In this study, we investigated the antibacterial properties of PTS and PTSO, two organosulfur derivatives of Allium cepa.These findings provide initial insights into the potential of both Allium species for further investigation in veterinary practice.However, in vivo trials are required to evaluate their efficacy in dogs.

Table 1 .
Resistance percentages of Staphyloccocus pseudintermedius containing the mecA gene to Allium extracts and antibiotics.

Table 2 .
Comparison of percentages of Staphylococcus psedintermedius strains sensitive to PTS/PTSO and resistant to the other antibiotics tested.
Turnidge and Paterson (2007) excluded due to the presence of the mecA gene.Colistin was not tested because it has no effect against Gram-positive bacteria.Sensitivity and resistance are classified using a previously established MIC cut-off, followingTurnidge and Paterson (2007)

Table 3 .
Percentages of antibiotic and PTS/PTSO resistance in ESBL-Enterobacteriaceae.

Table 4 .
Comparison of percentages of Enterobacteriaceae strains sensitive to PTS/PTSO and resistant to the antibiotics tested.