A Paternal Fish Oil Diet Preconception Modulates the Gut Microbiome and Attenuates Necrotizing Enterocolitis in Neonatal Mice

Epidemiology and animal studies suggest that a paternal history of toxicant exposure contributes to the developmental origins of health and disease. Using a mouse model, our laboratory previously reported that a paternal history of in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased his offspring’s risk of developing necrotizing enterocolitis (NEC). Additionally, our group and others have found that formula supplementation also increases the risk of NEC in both humans and mice. Our murine studies revealed that intervening with a paternal fish oil diet preconception eliminated the TCDD-associated outcomes that are risk factors for NEC (e.g., intrauterine growth restriction, delayed postnatal growth, and preterm birth). However, the efficacy of a paternal fish oil diet in eliminating the risk of disease development in his offspring was not investigated. Herein, reproductive-age male mice exposed to TCDD in utero were weaned to a standard or fish oil diet for one full cycle of spermatogenesis, then mated to age-matched unexposed females. Their offspring were randomized to a strict maternal milk diet or a supplemental formula diet from postnatal days 7–10. Offspring colon contents and intestines were collected to determine the onset of gut dysbiosis and NEC. We found that a paternal fish oil diet preconception reduced his offspring’s risk of toxicant-driven NEC, which was associated with a decrease in the relative abundance of the Firmicutes phylum, but an increase in the relative abundance of the Negativicutes class.

Introduction
The Developmental Origins of Health and Disease (DOHaD) concept suggests that the intrauterine environment has a significant impact on adult health. In the past, maternal factors were the target of many DOHaD studies, with only a few investigating the role of the father. This may be because the father's sole contribution to pregnancy is his seminal fluid, which historically had not been theorized to play a large role in neonatal health. Recently, we have discovered that seminal fluid contributes to DOHaD, which means that paternal preconception diet and habits may influence the outcomes of not only his partner's pregnancy but also their offspring's postnatal health.
Seminal fluid contains spermatozoa, nutrients, and microbes [1]. Our understanding of the transfer of sexually transmitted infections from male to female suggests a role for semen in influencing the vaginal and intrauterine microbiome [1]. Mandar et al. reported an association between males with genital tract inflammation and the predominance of Gardnerella vaginalis in their partners [2]; a bacterium that has been linked to poor pregnancy outcomes (e.g., preterm birth and chorioamnionitis) and has the potential to promote the onset of diseases of prematurity [3]. Seminal fluid has also been reported to influence conditions such as preeclampsia through the induction of immune tolerance in pregnant partners [1]. Together, these studies suggest that, in theory, mixing of the endometrial and seminal microbiome during conception could influence maternal-embryonic interactions during implantation and placentation, ultimately impacting offspring development [4].
Our laboratory previously reported that a history of in utero toxicant exposure reduced sperm quality in male mice and contributed to DOHaD in his offspring through placental dysfunction in his unexposed partner. This placental dysfunction translated to an increased risk of preterm birth and intrauterine growth restriction among their offspring [5]. We also reported that these offspring were susceptible to necrotizing enterocolitis (NEC), an inflammatory intestinal disease characterized by gut dysbiosis that impacts approximately 1 to 3 of every 1000 live births in North America. NEC is characterized by two forms of gut bacterial dysbiosis: Firmicute and Proteobacteria dysbiosis [6,7]. Infants with Firmicute dysbiosis typically develop NEC earlier than infants who experience Proteobacteria dysbiosis. Recent studies also suggest that infants susceptible to NEC present with increased abundances of Gammaproteobacteria, but decreased abundances of Negativicutes [8]. However, unlike Proteobacteria dysbiosis, Firmicute dysbiosis also affects approximately 10% of healthy infants, who do not develop NEC [7]. Hence, it is of importance to identify differences in the presentation of Firmicute dysbiosis in healthy infants versus those who are known to be susceptible to NEC. Additionally, it has been reported that a neonatal diet (e.g., formula feeding vs. maternal milk) also impacts the onset of gut dysbiosis in neonates [9,10]. It should also be noted that formula feeding is a risk factor for NEC in neonates [11].
Our group reported that intervening with a fish oil diet preconception in males with a history of toxicant exposure prevented preterm birth and intrauterine growth restriction in their offspring in association with improved placental function [12]. Neonatal development of offspring was also enhanced [13]. We hypothesized that this intervention would also reduce the risk of NEC since preterm birth and intrauterine growth restriction are independent risk factors for this disease. Therefore, in the present study, we aimed to determine if a paternal fish oil diet preconception influences early-life microbial seeding in his offspring and if this difference in microbial seeding is associated with a reduced risk of toxicant-driven NEC. We also examined the effect of postnatal formula supplementation since this diet has been linked to gut dysbiosis and the onset of NEC in human infants.
We report that a paternal fish oil diet preconception altered Firmicute abundance in the gut of his offspring. A preconception fish oil diet increased the relative abundance of Negativicutes, a class of Firmicutes that are typically reduced in human infants with NEC [14]. Additionally, a preconception fish oil diet improved intestinal histology and reduced the risk of toxicant-driven NEC in offspring. To our understanding, we are the first to report a potential relationship between a father's preconception diet, early-life microbial seeding, and the risk of NEC in his offspring.

Paternal Preconception Diet Influences His Offspring's Gut Microbial Diversity at the Phylum Level in F2 CT Pups
A major goal of our study was to determine whether paternal preconception diet or offspring postnatal diet influences bacterial diversity in the intestines of neonatal mice. Male C57BL/6 pups were weaned to a standard or a fish oil-supplemented diet for 7 weeks, then Mar. Drugs 2022, 20, 390 3 of 17 mated to age-matched C57BL/6 females maintained on the standard-diet preconception, during gestation, and postpartum. Offspring remained with the dam and were randomized to a strict maternal milk diet or a supplemental formula diet from postnatal day (PND) 7 through 10. On PND 11, the colon contents of offspring were collected and processed using Next-Generation Sequencing; pup nomenclature can be found in Table 1. When compared to maternal milk-fed pups sired by a father who received a standard diet (F2 CT pups), we found their formula supplemented counterparts, sired by standard diet fathers (F2 CT /Form pups), exhibited similar Shannon Diversity Indexes (p = 0.9498). Compared to F2 CT pups, a paternal fish oil diet preconception led to a non-significant increase in the Shannon Diversity Index of maternal milk-fed pups (F2 CT /Fish pups) (p = 0.1760) and formula supplemented offspring (F2 CT /Fish/Form pups) (p = 0.3136) ( Figure 1A). F2 CT /Fish pups also exhibited a non-significant increase in the relative abundance of Proteobacteria (p = 0.2706), like the F2 CT /Fish/Form pups (p = 0.5708). F2 CT /Fish pups also displayed a small decrease in the relative abundance of Firmicutes (p = 0.2889), like the F2Fish/Form pups (p = 0.4658), but these trends did not reach significance ( Figure 1B).

Paternal Diet Preconception Influences the Relative Abundance of Firmicute Classes in F2 CT Pups
Firmicute dysbiosis is suggested to be a biomarker of necrotizing enterocolitis (NEC) [6]; therefore, we examined how paternal and neonatal diet influenced classes of bacteria within the Firmicute phylum (i.e., Bacilli, Clostridia, Negativicutes, and Tissierellia). The relative abundance of Bacilli was similar between F2 CT and F2 CT /Form pups (p = 0.9958). F2 CT /Fish pups displayed a significant decrease in the relative abundance of Bacilli (p = 0.0014); however, F2 CT/ Fish/Form pups exhibited similar relative abundances of Bacilli (p = 0.1968) when compared to F2 CT pups ( Figure 2A). F2 CT /Fish pups also displayed a small increase in the relative abundance of Clostridia (p = 0.0573). F2 CT /Fish/Form pups exhibited a relative abundance of Clostridia that was like the F2 CT pups (p = 0.9670) ( Figure 2B). Significantly, the relative abundance of Negativicutes was increased in F2 CT /Fish (p = 0.0025) and F2 CT /Fish/Form offspring (p = 0.0160) ( Figure 2C). There was no difference in the relative abundance of Tissierellia between groups ( Figure 2D).

Paternal Diet Preconception Influences the Relative Abundance of Bacilli and Negativicutes Orders in F2 CT Pups
Since a paternal fish oil diet led to a significant decrease in the relative abundance of Bacilli, we measured the relative abundances of bacterial groups within this class. We found that a paternal fish oil diet preconception and/or neonatal formula supplementation did not have a significant impact on the relative abundance of bacterial groups within the orders of Bacillales (p ≥ 0.5869) and Lactobacillales (p ≥ 0.0652) ( Figure 3A,B). Since a paternal fish oil diet led to a significant increase in the relative abundance of Negativicutes ( Figure 2C), herein, we also measured the relative abundances of bacterial groups within this class. We found that compared to pups of fathers maintained on the standard diet, a paternal fish oil diet preconception was associated with a significant decrease in the relative abundance of the order Selenomondales in F2 CT /Fish and F2 CT /Fish/Form pups (p < 0.0001). The relative abundance of the Veillonellaceae family was also significantly decreased in F2 CT /Fish (p < 0.0001) and F2 CT /Fish/Form offspring (p < 0.0001) compared to F2 CT offspring ( Figure 4A).

Paternal Diet Preconception Influences the Relative Abundance of Firmicute Classes i Pups
Firmicute dysbiosis is suggested to be a biomarker of necrotizing enterocolitis [6]; therefore, we examined how paternal and neonatal diet influenced classes of b within the Firmicute phylum (i.e., Bacilli, Clostridia, Negativicutes, and Tissierelli relative abundance of Bacilli was similar between F2CT and F2CT/Form pups (p = 0 F2CT/Fish pups displayed a significant decrease in the relative abundance of Baci increase in the relative abundance of Clostridia (p = 0.0573). F2CT/Fish/Form pups exhibited a relative abundance of Clostridia that was like the F2CT pups (p = 0.9670) ( Figure 2B). Significantly, the relative abundance of Negativicutes was increased in F2CT/Fish (p = 0.0025) and F2CT/Fish/Form offspring (p = 0.0160) ( Figure 2C). There was no difference in the relative abundance of Tissierellia between groups ( Figure 2D).

Paternal Diet Preconception Influences the Relative Abundance of Bacilli and Negativicutes Orders in F2CT Pups
Since a paternal fish oil diet led to a significant decrease in the relative abundance of Bacilli, we measured the relative abundances of bacterial groups within this class. We found that a paternal fish oil diet preconception and/or neonatal formula supplementation did not have a significant impact on the relative abundance of bacterial groups within the orders of Bacillales (p ≥ 0.5869) and Lactobacillales (p ≥ 0.0652) ( Figure 3A,B). Since a paternal fish oil diet led to a significant increase in the relative abundance of Negativicutes ( Figure 2C), herein, we also measured the relative abundances of bacterial groups within this class. We found that compared to pups of fathers maintained on the standard diet, a paternal fish oil diet preconception was associated with a significant decrease in the relative abundance of the order Selenomondales in F2CT/Fish and F2CT/Fish/Form pups (p < 0.0001). The relative abundance of the Veillonellaceae family was also significantly decreased in F2CT/Fish (p < 0.0001) and F2CT/Fish/Form offspring (p < 0.0001) compared to F2CT offspring ( Figure 4A).   We further investigated how paternal and neonatal diets influenced the relative abundances of bacterial groups classified under the order of Selenomondales and the family of Veillonellaceae. We found that paternal and neonatal diets did not greatly impact the relative abundances of Sporomusaceae (p ≥ 0.7805) or Selenomonadaceae (p ≥ 0.7805) ( Figure  4B). We also report that, compared to F2CT pups, F2CT/Fish and F2CT/Fish/Form pups displayed significant reductions in the relative abundances of Veillonella (p < 0.0001) and Megasphaera (p ≥ 0.0007). F2CT/Fish and F2CT/Fish/Form pups also displayed a significant increase in the relative abundance of Negativicoccus massiliensis (p < 0.0001) compared to F2CT pups. F2CT/Form pups displayed no significant difference in the relative abundances We further investigated how paternal and neonatal diets influenced the relative abundances of bacterial groups classified under the order of Selenomondales and the family of Veillonellaceae. We found that paternal and neonatal diets did not greatly impact the relative abundances of Sporomusaceae (p ≥ 0.7805) or Selenomonadaceae (p ≥ 0.7805) ( Figure 4B). We also report that, compared to F2 CT pups, F2 CT /Fish and F2 CT /Fish/Form pups displayed significant reductions in the relative abundances of Veillonella (p < 0.0001) and Megasphaera (p ≥ 0.0007). F2 CT /Fish and F2 CT /Fish/Form pups also displayed a significant increase in the relative abundance of Negativicoccus massiliensis (p < 0.0001) compared to F2 CT pups. F2 CT /Form pups displayed no significant difference in the relative abundances of Veillonella, Megasphaera, or Negativicoccus massiliensis (p ≥ 0.1607) ( Figure 4C) compared to F2 CT pups.

Paternal Diet Preconception Alters the Firmicute Abundance in F2 TCDD Pups
As described above, we observed that a paternal fish oil diet preconception was associated with a small increase in offspring Shannon Diversity Index as well as an altered relative abundance of Negativicutes and associated bacterial groups. An increased Shannon Diversity Index and an increase in the presence of Negativicutes are associated with a reduced risk of developing NEC in human infants [6,8,15,16]. Therefore, our next goal was to employ our toxicant-driven murine model of NEC to determine if intervening with a paternal fish oil diet preconception improves NEC outcomes by influencing microbial diversity in offspring. In this model, the offspring of males with a history of in utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure are susceptible to NEC and postnatal formula supplementation further increases their risk of disease [17].
We report no significant difference in the Shannon Diversity index of maternal milkfed pups sired by standard diet fathers with a history of TCDD exposure (F2 TCDD pups) when compared to F2 CT pups. However, formula supplemented pups sired by standard diet fathers with a history of TCDD exposure (F2 TCDD /Form pups) exhibited a significant decrease in their Shannon Diversity Index compared to F2 TCDD pups (p = 0.0384) ( Figure 5A). Next, we observed the relative abundance of Firmicute classes among pups with a history of TCDD exposure. When compared to F2 CT pups, maternal milk fed pups sired by fathers with a history of TCDD exposure who received fish oil (F2 TCDD /Fish pups) displayed a significant decrease in their relative abundance of Bacilli (p = 0.0002). Additionally, F2 TCDD /Fish pups exhibited a significant increase in their relative abundance of Negativicutes compared to F2 CT pups (p = 0.0205). These trends persisted in formula supplemented pups sired by fathers with a history of TCDD exposure who received fish oil (F2 TCDD /Fish/Form pups), but did not reach significance. We report no significant differences in the relative abundances of Clostridia and Tissierellia between groups. However, F2 TCDD /Fish pups (p < 0.0001), and F2 TCDD /Fish/Form pups (p = 0.0082) displayed a significant reduction in the relative abundance of Bacilli compared to F2 TCDD pups. F2 TCDD /Fish pups also exhibited a significant increase in the relative abundance of Negativicutes when compared to F2 TCDD pups (p = 0.0185). However, this trend did not reach significance in F2 TCDD /Fish/Form pups (p = 0.0543) ( Figure 5B).

Paternal Fish Oil Consumption Alters the Abundance of Negativicutes in F2 TCDD Pups
Next, we examined whether a paternal fish oil diet also increased the relative abundance of bacterial groups within the class of Negativicutes in pups with a history of TCDD exposure. F2 TCDD /Fish and F2 TCDD /Fish/Form pups exhibited a significant increase in the relative abundances of Selenomondales and Veillonellaceae when compared to F2 CT pups (p < 0.0001) ( Figure 6A). Like what was demonstrated by F2 CT /Fish pups, a paternal fish oil diet preconception did not affect the relative abundances of Sporomusacae and Selenomonadaceae in pups with a history of TCDD exposure ( Figure 6B). F2 TCDD pups exhibited a significant increase in the relative abundance of Megaspahera (p = 0.0302), but no differences in the relative abundance of Veillonella, Negativicoccus massilienis or Dialister sp. Marseille-P5638 when compared to F2 CT pups. F2 TCDD /Form pups exhibited similar abundances of Veillonella, Megasphaera, Negativicoccus massilienisis, and Dialaister sp. Marseillie-P5638 when compared to F2 CT pups. F2 TCDD /Fish and F2 TCDD /Fish/Form pups exhibited a significant decrease in their relative abundance of Veillonella and Megasphaera (p < 0.0001), but a significant increase in Negativicoccus massiliensis (p < 0.0001) when compared to F2 CT pups ( Figure 6C). Additionally, F2 TCDD /Fish and F2 TCDD /Fish/Form pups exhibited a significant decrease in the relative abundance of Selenomondales and Veillonellaceae when compared to F2 TCDD pups (p < 0.0001) (Supplementary Figure S1A). Significantly, F2 TCDD /Fish and F2 TCDD /Fish/Form pups displayed a decrease in Veillonella and Megaspahera, but an overall increase in Negativicoccus massiliensis (p < 0.0001) when compared to F2 TCDD pups (Supplementary Figure S1B).

Paternal Fish Oil Consumption Alters the Abundance of Negativicutes in F2TCDD Pups
Next, we examined whether a paternal fish oil diet also increased the relative a significant increase in Negativicoccus massiliensis (p < 0.0001) when compared to F2CT pups ( Figure 6C). Additionally, F2TCDD/Fish and F2TCDD/Fish/Form pups exhibited a significant decrease in the relative abundance of Selenomondales and Veillonellaceae when compared to F2TCDD pups (p < 0.0001) (Supplementary Figure S1A). Significantly, F2TCDD/Fish and F2TCDD/Fish/Form pups displayed a decrease in Veillonella and Megaspahera, but an overall increase in Negativicoccus massiliensis (p < 0.0001) when compared to F2TCDD pups (Supplementary Figure S1B).

A Paternal Fish Oil Diet Preconception Attenuates Susceptibility to NEC
Our final aim was to determine if a paternal fish oil diet preconception reduced the risk of NEC in his offspring. F2 CT and F2 CT /Fish pups exhibited healthy intestines, denoted by elongated enterocytes, little-to-no sloughing of the intestinal crypt from the villi, and integrity of the submucosa ( Figure 7A,C). Additionally, F2 CT and F2 CT /Fish pups did not develop NEC ( Figure 7B and Table 2). F2 CT /Form pups displayed mild intestinal sloughing ( Figure 7B) and 40% of these pups developed low-grade NEC ( Figure 7I and Table 2). Limited intestinal sloughing was observed in F2 CT /Fish/Form pups ( Figure 7D) and the incidence of NEC was reduced to 20% in this group ( Figure 7I and Table 2). F2TCDD pups exhibited severe intestinal sloughing and impaired submucosa integrity compared to F2CT pups and this trend persisted in F2TCDD/Form pups ( Figure 7E,F). However, F2TCDD/Fish and F2TCDD/Fish/Form pups displayed improved intestinal integrity and enterocyte elongation. F2TCDD/Fish and F2TCDD/Fish/Form pups also exhibited a reduction in the separation of intestinal crypt and villi ( Figure 7G,H). This improvement in intestinal histology among F2TCDD/Fish and F2TCDD/Fish/Form pups translated to an attenuated risk for the development of NEC. F2TCDD pups (p = 0.0292) and F2TCDD/Form pups (p = 0.0407) had the highest incidences of NEC at 66% and 83%, but the risk of NEC was eliminated in F2TCDD/Fish pups and reduced to 58% in F2TCDD/Fish/Form pups ( Figure 7I and Table 2).  F2 TCDD pups exhibited severe intestinal sloughing and impaired submucosa integrity compared to F2 CT pups and this trend persisted in F2 TCDD /Form pups ( Figure 7E,F). However, F2 TCDD /Fish and F2 TCDD /Fish/Form pups displayed improved intestinal integrity and enterocyte elongation. F2 TCDD /Fish and F2 TCDD /Fish/Form pups also exhibited a reduction in the separation of intestinal crypt and villi ( Figure 7G,H). This improvement in intestinal histology among F2 TCDD /Fish and F2 TCDD /Fish/Form pups translated to an attenuated risk for the development of NEC. F2 TCDD pups (p = 0.0292) and F2 TCDD /Form pups (p = 0.0407) had the highest incidences of NEC at 66% and 83%, but the risk of NEC was eliminated in F2 TCDD /Fish pups and reduced to 58% in F2 TCDD /Fish/Form pups ( Figure 7I and Table 2). Overall, a paternal fish oil diet preconception reduced the risk of NEC in his offspring. Interestingly, offspring sired by fathers who received a fish oil diet preconception displayed similarities in their Firmicute abundances independent of the father's history of TCDD exposure (Table 3). This suggests that Firmicute dysbiosis may have a similar phenotype in healthy versus unhealthy infants and the onset of NEC in infants with Firmicute dysbiosis is also driven by additional risk factors such as prematurity, formula supplementation, and inflammation. Table 3. Patterns in Firmicute abundances among F2 CT and F2 TCDD pups based on their father's preconception diet.

Paternal Standard Diet Paternal Fish Oil Diet
The arrows reference an increase or decrease in bacterial abundances in pups as it relates to their father's preconception diet.

Discussion
Necrotizing enterocolitis (NEC) is a rare, but serious, intestinal inflammatory disease. Although NEC most commonly affects infants that were born preterm, up to 10% of babies with this condition are delivered at term [18]. Some groups have also reported that mortality rates associated with NEC are similar among term and preterm infants [19]. Both term and preterm infants can exhibit characteristics, such as Firmicute dysbiosis, which have been linked to NEC. Thus, it is important to identify factors that influence microbial seeding in early life and determine whether these factors influence the onset of NEC. Herein, our major goals were to determine the impact that paternal preconception diet and offspring postnatal diet had on early-life microbiome composition, determine whether a father's history of toxicant exposure impacted his offspring's intestinal microbiome, and, finally, determine if any of these parameters were associated with the onset of NEC.
To determine whether paternal or neonatal diet influenced microbial diversity in F2 CT mice, we provided their fathers with a fish oil diet preconception. We previously reported that this diet influenced multiple sperm parameters, which positively impacted placental function, pregnancy outcomes, and offspring development [12]. Our findings, presented herein, demonstrate that this diet slightly increased microbial diversity in F2 CT /Fish and F2 CT /Fish/Form pups ( Figure 1A). Furthermore, F2 CT /Fish and F2Fish/Form pups displayed a reduction in their relative abundance of Firmicutes ( Figure 1B). These data support the theory that priming of neonatal microbial seeding may begin in utero [20]. It should be noted that our findings are in alignment with a murine study conducted by Yu et al. suggesting that direct fish oil treatment decreased the relative abundance of the Firmicute phylum [21]. Additional studies from others have suggested that the omega-3 fatty acids found in fish oil can influence the gut microbiome by modulating the growth of opportunistic bacteria [22].
Next, we aimed to identify differences in the relative abundances of bacterial classes within the Firmicute phylum to observe characteristics of dysbiosis. We reported that a paternal fish oil diet preconception had a more significant impact on these parameters than a neonatal diet. F2 CT /Fish pups displayed a significant reduction in their relative abundance of Bacilli (Figure 2A), but a non-significant increase in their relative abundances of Clostridia ( Figure 2B). Additionally, F2 CT /Fish and F2 CT /Fish/Form pups exhibited a significant increase in their relative abundance of Negativicutes ( Figure 2C); however, the relative abundance of Tissierellia was not impacted ( Figure 2D). These findings confirm that fish oil can influence the relative abundance of Bacilli, a concept that was previously reported by Curone et al. using a rabbit model [23]. Unlike our findings, other groups have reported that fish oil reduces the abundance of Clostridia in humans and mice [24,25]suggesting that this may be the case when fish oil is provided to the subject directly but not when the supplement is provided indirectly as in our study.
Since a paternal fish oil diet led to a significant decrease in the relative abundance of Bacilli, we further investigated the relative abundances of Bacialles ( Figure 3A) and Lactobacialles ( Figure 3B) but found no differences between the groups. Likely because these bacterial groups are theorized to be transferred from mother to infant during birth and maternal feeding [26,27]. Others have reported that Bacilli abundance is increased following neonatal formula supplementation [28]; although, our data support this finding, the trend did not reach significance (Figures 2 and 3A,B).
Our next aim was to investigate the relationship between paternal and neonatal diet and the relative abundance of Negativicutes in the gut of offspring. A study conducted by Hosomi et al. suggests that the dietary fish oil fatty acids eicosapentanoic acid and docosahexaenoic acid increased the relative abundance of Negativicutes in the intestines of rats who were provided the diet directly [29]. Supporting the study conducted by Hosomi et al., we report that F2 CT /Fish and F2 CT /Fish/Form pups had a significantly increased relative abundance of Negativicutes ( Figure 2C), which was associated with a significant increase in the abundance of Veillonellaceae and a significant decrease in the abundance of Selenomondales ( Figure 4A). Paternal and neonatal diet did not influence families within the order of Selenomondales ( Figure 4B); however, F2 CT /Fish and F2 CT /Fish/Form pups exhibited reduced abundances of Veillonella and Megashpaera but increased abundances of Negativicoccus massiliensis ( Figure 4C). To our knowledge, we are the first group to examine the relationship between the abundance of Negativicutes in offspring and paternal diet preconception.
Our final aim was to determine if a paternal fish oil diet preconception could reduce the risk of his offspring developing toxicant-driven NEC in association with modulation of the gut microbiome. A reduction in microbial diversity, marked by a decrease in the Shannon Diversity Index is a characteristic of NEC [30] and we report that F2 TCDD /Form pups exhibited a significant decrease in their Shannon Diversity Index compared to F2 TCDD pups ( Figure 5A). This supports our previous report that formula supplementation increases the risk of NEC in pups with a history of toxicant exposure [17]. Additionally, F2 TCDD pups displayed an over-growth of Bacilli but F2 TCDD /Fish pups displayed a reduction in their relative abundance of Bacilli, but an increase in the relative abundance of Negativicutes, respectively ( Figure 5B).
It should be noted that the absence of Negativicutes in the intestines of neonates has been linked to the development of NEC in humans [8]. F2 TCDD /Fish pups and F2 TCDD /Fish/Form pups displayed a significant increase in their relative abundance of Negativicutes. Similar to F2 CT /Fish pups, F2 TCDD /Fish and F2 TCDD /Fish/Form pups had a significantly increased relative abundance of Veillonellaceae, but a significantly decreased relative abundance of Selenomondales ( Figure 6A). Additionally, the trend of a paternal fish oil diet having no impact on the relative abundance of bacterial members within Selenomondales persisted ( Figure 6B). Additionally, the trend of a paternal fish oil diet decreasing the relative abundance of Veillonella and Megaspahera, while significantly increasing the relative abundance of Negativicoccus massiliensis also persisted in F2 TCDD /Fish and F2 TCDD /Fish/Form pups ( Figure 6C).
In this study, we confirmed our previous report that a history of paternal toxicant exposure and postnatal formula supplementation are independent risk factors for NEC [17]. Herein, 66% of F2 TCDD pups and 83% of F2 TCDD /Form pups developed NEC; whereas 0% of F2 CT pups and 40% of F2 CT /Form pups developed NEC. The risk of NEC was eliminated in F2 CT /Fish pups and reduced to 20% in F2 CT /Fish/Form pups. This trend persisted in pups with a history of toxicant exposure, as a fish oil intervention reduced their incidence of NEC to 0% in F2 TCDD /Fish pups and 25% in F2 TCDD /Fish/Form pups (Table 2). Additionally, F2 CT /Form pups exhibited intestinal sloughing; however, this sloughing was more severe and accompanied by impaired intestinal integrity in the F2 TCDD and F2 TCDD /Form pups ( Figure 7A). Although some F2 CT /Form pups developed NEC, these incidences did not reach significance, unlike F2 TCDD and F2 TCDD /Form pups ( Figure 7B).
Overall, our data suggest that a paternal fish oil diet preconception may have a similar effect on microbial seeding in F2 CT neonatal mice and F2 TCDD neonatal mice (Table 3). Our findings also suggest that formula supplementation and a history of paternal TCDD exposure are independent risk factors for NEC in neonatal mice. A paternal fish oil diet eliminated the risk of NEC in maternal milk fed offspring with a history of toxicant exposure and reduced the risk of disease in formula supplemented neonatal mice. Our data suggest that a paternal fish oil diet preconception helps reduce the risk of NEC by modulating the neonatal gut microbiome; however, our findings also suggest that additional factors (e.g., prematurity, inflammation, and formula supplementation) drive NEC outcomes as well.
We previously established that the mothers of pups sired by fathers with a history of TCDD exposure exhibit poor placental function, which put their offspring at risk of early-life complications. As has been demonstrated by us and others, the placenta is heavily influenced by the father and plays significant roles in fetal and neonatal development. We also reported that the mothers of F2 TCDD /Fish pups had improved placental function and their offspring were less susceptible to early-life conditions, likely because this paternal dietary intervention improved sperm function, which translated to improved placental function [12]. Additionally, the placental phenotype has been suggested to contribute to the development of the infant gut microbiome as a consequence of fetal ingestion of amniotic fluid [31][32][33]. However, to our knowledge, ours is the first study to explore the neonatal microbiome following toxicant exposure of the father. Our study also suggests that the father's preconception diet may also influence the microbial landscape of his offspring, potentially due to changes in placental function. However, it should be noted that the current study used a small sample size of three pups per group from microbial analyses; therefore, larger group sizes are needed to confirm the trends that we have reported. Additionally, due to the non-invasive nature of this study, it would also be worthwhile to measure these outcomes using an epidemiological study design.

Animals
Adult (10-12 weeks) and neonatal C57BL/6 mice were used in this study. Adult mice were obtained from Envigo (Indianapolis, IN, USA) or born in-house. All neonatal mice were born in-house. Animals were housed in the Barrier Animal Care Facility at Vanderbilt University Medical Center, which is free of common mouse pathogens. Adult mice were provided food and water ad libitum. Animal room temperatures were maintained between 22 and 24 • C and relative humidity of 40-50% on a 12 h light:dark schedule. Experiments described in this study were approved by Vanderbilt University's Institutional Animal Care and Use Committee (IACUC) per the Animal Welfare Act protocol #M2000098.

Mating, Exposure, and Diet Scheme
Virgin 10 to 12-week-old C57BL/6 females were mated with intact males of similar age. Females were weighed daily and monitored for the presence of a vaginal semen plug; denoting copulation had occurred. The morning a vaginal plug was identified, the F0 dam was considered pregnant (denoted as embryonic day 0.5 (E0.5)) and moved to a new cage. F1 offspring were typically born on E20 and weaned at 3 weeks.

TCDD Exposure
F0 dams were exposed to 10 µg/kg of TCDD in corn oil or corn oil vehicle by gavage on E15.5 as previously described [13,17].

Diet and Mating Scheme for the F1 Generation
Purina Mills (TestDiet division) provided the 5% Menhaden fish oil diet, which also contained 1.5% corn oil to prevent depletion of omega-6 fatty acids. Menhaden fish oil, (OmegaProtein, Houston, TX, USA) has an established fatty acid profile (~40% omega-3 fatty acids) and was processed to remove dioxins and polychlorinated biphenyls. The fish oil diet is a modification of Purina's low phytoestrogen rodent chow, 5VR5, which was used as the control (standard) diet. Both diets are matched for energy content, but the fish oil diet had increased fat content with slightly lower protein and carbohydrate content. The fish oil diet was maintained in vacuum-sealed bags at (−20 • C) until use and once provided to mice, replaced every 3 days.
F1 males were weaned for 7 weeks on a fish oil or standard diet (one full cycle of spermatogenesis) and mated at 10-12 weeks of age with age-matched unexposed C57BL/6 females. Once a vaginal semen plug was identified, dams were singly housed until parturition. F2 offspring were typically born between E18.5 and 20.

Formula Feeding
As previously described [13,17], beginning on postnatal day 7 (PND7), pups were weighed, and randomized to a strict maternal milk diet or a supplemental formula diet. Pups were bottle-fed 30 µL of freshly prepared Esbilac formula diluted in autoclaved cage water three times a day over the course of four days using a small nipple attached to a 1 ml syringe (Miracle Nipple Mini for Pets and Wildlife). Each 30 µL dose was provided in two aliquots of 15 µL, each 10 min apart. All pups remained with dams for the duration of the study and were allowed to nurse ad libitum.

Euthanasia and Sample Collection
On PND11 at 1100 h local time, pups were weighed, then euthanized by decapitation performed under deep anesthesia per AAALAC guidelines. Following euthanasia, the peritoneal cavity was opened, and the intestines were identified. The colon was then excised from the small intestine and rectum. Upon removal of the colon, sterile tools were used to push the colon contents out of the colon and into a sterile tube. Colon contents were stored at −80 • C until further use. The remainder of the intestines was collected and prepared for histological analyses as previously described [17].

Bacterial Isolation and Next-Generation Sequencing
The Qiagen Power Soil Kit (Germantown, MD, USA) was used to isolate bacterial species within colon contents samples. Vanderbilt's Microbiome Core sequenced the bacterial populations within each sample using 16s-based Next-Generation sequencing. Bacterial abundances were determined by visualizing and extrapolating sequence data from Krona Charts on the Illumina platform. Krona Chart data included the Shannon diversity index of each pup, as well as their relative abundances of various microbial populations. Extrapolated data from QIIME's Krona Chart have previously been used in multidimensional analyses of the estimated relative abundances of microbes in a sample [34][35][36].

Assessment of Necrotizing Enterocolitis
Intestinal tissues were subjected to hematoxylin and eosin staining, and a histologic injury score was used to assess the development of Necrotizing Enterocolitis, as previously described [17]. A grade of 0-1 was considered healthy, whereas a grade of two or more was considered NEC; increasing scores indicate increased severity. Three blinded scorers were provided two images per pup and asked to determine an NEC score for each image and the scores were averaged. Three averaged scores for each pup were used to determine one NEC score per pup.

Statistical Analysis
Group results were compared for significance using Prism's one-way ANOVA and the Tukey post hoc test. For all microbial analyses, three non-littermates were used to obtain the average for each group. For histological analyses, 5-8 non-littermates were used per group. The presented images are representative of each group. The data are represented as the mean ± standard deviation and p < 0.05 was considered significant. Significance was determined by comparing each treatment group to F2 CT pups. In each group, approximately half of the pups were male and the other half were female. The majority of the pups were male in groups with uneven sample sizes. It should be noted that a major limitation of the current study was the small group sizes used for microbial analyses, due to the number of variables that were measured.

Conclusions
Herein, we report that a paternal fish oil diet preconception influences early-life microbial seeding in the intestines of their offspring by increasing the relative abundance of Negativicutes. Pups with a paternal history of TCDD exposure were susceptible to NEC and this susceptibility was associated with an increase in the relative abundance of Megashpaera. Intervening with a paternal fish oil diet preconception significantly reduced the relative abundance of Veillonella and Megasphaera, but significantly increased the relative abundance of Negativicoccus massiliensis. This suggests that a paternal fish oil diet may reduce the incidence of NEC in pups with a history of toxicant exposure by influencing the relative abundances of Veillonella, Megasphaera, and Negtivicoccus massiliensis (types of Negativicutes) within the gut of their offspring. It should be noted that pups sired by unexposed fathers maintained on the standard diet exhibited a similar Firmicute diversity compared to pups susceptible to NEC, suggesting that factors other than gut dysbiosis, such as prematurity and inflammation, also drive disease development and outcomes. A limitation of this study was using an n of three for the Next Generation Sequence analyses due to the number of variables we assessed. Hence, this study should be repeated with a larger sample size and in other animal models of NEC to validate our current findings.


s2fieldsofstudy: Biology Medicine

text:
Antimicrobial potency, prevention ability, and killing efficacy of daptomycin-loaded versus vancomycin-loaded β-tricalcium phosphate/calcium sulfate for methicillin-resistant Staphylococcus aureus biofilms

Growing evidence has shown that the efficacy of systemic administration of daptomycin for the treatment of methicillin-resistant Staphylococcus aureus (MRSA)-related infections is satisfactory. However, the clinical efficacy of the local administration of daptomycin for the management of osteoarticular infections remains unclear. This in vitro study compared the efficacy of daptomycin and vancomycin against MRSA biofilms. The elution kinetics of daptomycin and vancomycin, combined with gentamicin and loaded with either β-tricalcium phosphate/calcium sulfate or calcium sulfate, in the presence of MRSA infection, was assessed. Their efficacy in preventing biofilm formation and killing pre-formed biofilms was assessed using colony-forming unit count and confocal laser scanning microscopy. In addition, the efficacy of daptomycin, vancomycin, and gentamicin in prophylaxis and eradication of MRSA biofilms was also evaluated. Daptomycin + gentamicin and vancomycin + gentamicin displayed similar antimicrobial potency against MRSA, by either β-tricalcium phosphate/calcium sulfate or calcium sulfate. In the prevention assays, both daptomycin + gentamicin and vancomycin + gentamicin showed similar efficacy in preventing bacterial colony formation, with approximately 6 logs lower colony-forming units than those in the control group at both 1 and 3  days. The killing effect on pre-formed biofilms showed significant decreases of approximately 4 logs at 1 and 3  days following treatment with daptomycin + gentamicin and vancomycin + gentamicin. In addition, the confocal laser scanning microscopy results support the colony-forming unit data. Moreover, single use of vancomycin and gentamicin showed similar efficacies in preventing and killing MRSA biofilms, both of which were better than that of gentamicin. Our study demonstrated that vancomycin + gentamicin and daptomycin + gentamicin loaded with β-tricalcium phosphate/calcium sulfate or calcium sulfate showed similar prophylactic and killing effects on MRSA biofilms, implying a potential indication of local administration daptomycin for the treatment of MRSA-associated osteoarticular infections, especially if vancomycin administration presents limitations.

Growing evidence has shown that the efficacy of systemic administration of daptomycin for the treatment of methicillin-resistant Staphylococcus aureus (MRSA)-related infections is satisfactory. However, the clinical efficacy of the local administration of daptomycin for the management of osteoarticular infections remains unclear. This in vitro study compared the efficacy of daptomycin and vancomycin against MRSA biofilms. The elution kinetics of daptomycin and vancomycin, combined with gentamicin and loaded with either β-tricalcium phosphate/calcium sulfate or calcium sulfate, in the presence of MRSA infection, was assessed. Their efficacy in preventing biofilm formation and killing pre-formed biofilms was assessed using colony-forming unit count and confocal laser scanning microscopy. In addition, the efficacy of daptomycin, vancomycin, and gentamicin in prophylaxis and eradication of MRSA biofilms was also evaluated. Daptomycin + gentamicin and vancomycin + gentamicin displayed similar antimicrobial potency against MRSA, by either β-tricalcium phosphate/calcium sulfate or calcium sulfate. In the prevention assays, both daptomycin + gentamicin and vancomycin + gentamicin showed similar efficacy in preventing bacterial colony formation, with approximately 6 logs lower colony-forming units than those in the control group at both 1 and 3 days. The killing effect on pre-formed biofilms showed significant decreases of approximately 4 logs at 1 and 3 days following treatment with daptomycin + gentamicin and vancomycin + gentamicin. In addition, the confocal laser scanning microscopy results support the colony-forming unit data. Moreover, single use of vancomycin and gentamicin showed similar efficacies in preventing and killing MRSA biofilms, both of which were better than that of gentamicin. Our study demonstrated that vancomycin + gentamicin

Introduction
With the arrival of our aging society and an increasing number of patients with extremity fractures, the clinical use of artificial joints and internal fixations is increasing. The most challenging complications following joint replacement and fracture fixation procedures are implant-associated infections (IAIs), including periprosthetic joint infections (PJIs) and fracture-related infections (FRIs) (Trampuz and Widmer, 2006;Horton et al., 2021). According to recent studies, the cumulative incidence after primary hip or knee arthroplasty is 0.5% in the first year and 1.4% within 10 years, whereas the incidence of FRI ranges from 0.4 to 16.1%, with an average of 5%, and up to 30% following open fractures (Beam and Osmon, 2018;Foster et al., 2020;Jiang et al., 2021). Despite advances in surgical techniques, IAIs still pose great challenges to orthopedic surgeons, owing to their high risk of disability, mortality, and infection recurrence.
Staphylococcus aureus (S. aureus) is the main causative agent of IAIs, and methicillin-resistant S. aureus (MRSA) accounts for approximately 25-50% of the isolates (Gatti et al., 2022). They can form difficult-to-eradicate biofilms, which is the primary reason for infection recurrence (Arciola et al., 2018;Mandell et al., 2019). The frequently used strategy to remove bacterial biofilms is radical wound debridement with pulsed lavage (PL), followed by local implantation of antibiotics (Zimmerli, 2014;Depypere et al., 2020). PL is used to mechanically disrupt and eliminate bacterial biofilms in bones, soft tissues, and implant components. However, an in vitro study showed that PL could remove approximately 90% of the biofilms from artificial joint materials (Knecht et al., 2018). Therefore, in addition to physical removal, the local use of microbeads containing antibiotics is another effective way to control bacterial reproduction that may remain or subsequently enter the surgical site (Foster et al., 2020;Aggarwal et al., 2022). Materials that are commonly used to imbed the antibiotics are classified as non-absorbable [such as polymethyl methacrylate (PMMA)], or absorbable materials, [such as high-purity calcium sulfate (CS)]. Previous studies have indicated that they can be administered for both the treatment and prophylaxis of IAIs (Howlin et al., 2015;van Vugt et al., 2019;Aggarwal et al., 2022).
Initially, gentamicin (GEN, G) was locally administered to treat IAIs; however, with the increasing rate of antibiotic resistance, it was less frequently administered alone. Tobramycin (TOB, T) and vancomycin (VAN, V) are often administered for MRSArelated infections. However, increasing reports have indicated resistance to TOB (Zalavras et al., 2004;Jackson et al., 2021;Sumrall et al., 2021). Likewise, although VAN has been considered the "backbone" against MRSA-related infections, serious concerns have been raised regarding its limitations, such as the low therapeutic index, limited tissue penetration, slow bactericidal activity, as well as the increasing reports of resistance and treatment failure (Bruniera et al., 2015). For these reasons, the search for therapeutically effective alternative drugs, especially those that can replace their local use in IAIs, is critical (Stryjewski and Corey, 2009).
Many alternative drugs had been approved by the U.S. Food and Drug Administration (FDA) for the treatment of MRSArelated infections (Micek, 2007), such as linezolid, daptomycin (DAP, D), and tigecycline. DAP, a novel cyclic lipopeptide drug, was approved by the FDA in 2003 for the treatment of complicated skin and skin structure infections (cSSSIs), right-sided infective endocarditis, bacteremia following cSSSIs, and right-sided infective endocarditis. DAP has also been evaluated for IAIs treatment (Heidary et al., 2018). Several experimental studies comparing the efficacy of DAP and VAN have shown that DAP may have more advantages than VAN such as greater bone penetration and longer half-life. Most initial studies and clinical data proved that DAP is more efficacious than VAN in certain situations (e.g., with lower mortality and nephrotoxicity) for intravenous use in the treatment of bacteremia due to MRSA infections (Moenster et al., 2012;Moise et al., 2016). Further in vivo and in vitro experiments have demonstrated good outcomes of DAP in the treatment of osteomyelitis (Lamp et al., 2007). However, studies focusing on the local administration of DAP for the treatment of IAIs remain limited.
Concerning the local administration of antibiotics for the treatment of orthopedic infections, VAN in combination with GEN, loaded by carriers to achieve a persistent release effect, is more frequently selected clinically (Zalavras et al., 2004;Jackson et al., 2021). Thus, this study aimed to assess the efficiency of D + G and V + G loaded with two biomaterials, namely, β-tricalcium phosphate/calcium sulfate (β-TCP/CS) and CS, against bacterioplankton or biofilms of MRSA. One advantage of such antibiotic vehicles is that they are completely biodegradable, and thus do not require a second surgical removal. Additionally, Frontiers in Microbiology 03 frontiersin.org β-TCP/CS has an osteoconductive effect and provides bone scaffolding (Urban et al., 2007;Fernandez de Grado et al., 2018;Zhao et al., 2020). Here, we analyzed the release characteristics of DAP and compared the efficiency of DAP with that of VAN in preventing and killing MRSA biofilms, concerning a previously established in vitro biofilm model (Jiang et al., 2021). Through these experiments, we provide a theoretical basis for the local administration of DAP for the treatment of IAIs in clinical settings.

Materials and methods
Bacterial strains and growth media ATCC43300, an MRSA strain that has been shown to form biofilms on polymer surfaces, was selected for this study (Tan et al., 2012). The MRSA strain, provided by our laboratory, was cultured in trypsin-soybean broth (TSB; Pythonbio, Guangdong, China) overnight at 37°C with shaking at 200 rpm in shaking conditions. The minimum inhibitory concentrations (MICs) of D, V, and G were determined using the microfluidic dilution method (Wiegand et al., 2008).

Preparation of antibiotic-loaded beads (ALBs)
ALBs were prepared using Genex and Stimulan Rapid Cure products (Biocomposites Ltd., Staffordshire, United Kingdom). Genex is a synthetic biphasic material consisting of β-TCP and CS in a 1:1 weight ratio, whereas stimulant is a high-purity CS product. The mixing ratios for the D + G and V + G groups were 1,000 mg DAP (Hengrui Ltd., Jiangsu, China) with 1,000 mg GEN (Tianxin Ltd., Guangdong, China) 240 mg, and VAN (Eli Lily & Co., Indiana, United States) with gentamicin 240 mg, respectively, mixed with 10 ccs of Genex or Stimulan powder (Weiss et al., 2009;Kaplan et al., 2012). Similarly, the mix ratios for groups D, V, and G alone were DAP 1,000 mg, VAN 1,000 mg, and GEN 240 mg, with Genex or Stimulan powder per 10 ccs. A combination of sterile normal saline (NS), the respective antibiotic powder, and β-TCP/CS or CS powder was hand-mixed under aseptic conditions with dry powder and water for 30 s until a smooth paste was produced. The smooth paste was pressed in a flexible mold containing 4.8 mm wide hemispherical depressions placed at 20°C. After hardening for 24 h, the beads were released from the mold before use (Knecht et al., 2018).

Antibiotic-elution potency and duration for inhibition of planktonic bacteria
The modified Kirby-Bauer test (KBT) was used to determine the potency of ALBs over time (Boyle et al., 1973). Overnight cultures of MRSA (ATCC43300) were diluted to 1% in TSB medium, 100 μl of the diluted medium was applied to tryptic soy agar (TSA, Pythonbio, Guangdong, China) agar plates, and ALBs with D + G or V + G were placed centrally on top of the agar using aseptic forceps. Petri dishes were incubated at 37°C and 5% CO 2 for 24 h, followed by the analysis and recording of zones of inhibition (ZOIs). The beads were transferred to a new bacterial agar dish. The maximum diameter was measured every 24 h using a straightedge and recorded, and the process was repeated daily until the ZOIs measured diameter of less than 0.6 cm. Then, the ZOIs were calculated using the equation A = πr 2 , where A is the area of the circle, r is the radius (half of the diameter), and π is a constant (approximately equal to 3.142). This area is used instead of the diameter because the inhomogeneity of the beads sometimes leads to irregularly shaped ZOIs.

Prevention of biofilm formation and killing efficacy of established biofilms
ALBs and unloaded antibiotic beads (two beads per well) were placed in 12-well plates (Corning, NY, United States) and confocal dishes with a diameter of 35 mm (Corning, NY, United States). For biofilm prevention experiments, the bacteria were diluted to a concentration of approximately 10 6 colony-forming unit (CFU)/ ml and each well was inoculated with 2 ml of overnight culture and incubated at 37°C and 50 rpm on a shaker. The bacteria were incubated for 1 or 3 days, with the culture medium containing 2 ml of 10 6 CFU/ml of fresh bacteria, changed every 24 h. For the killing assay, 2 ml of 10 6 CFU/ml MRSA was inoculated per well and incubated at 37°C and 50 rpm on a shaker for 3 days to allow the establishment of biofilms, with 1 ml of the fresh medium changed daily. Then, two ALBs or unloaded antibiotic beads were added to each well with fresh medium, and the medium was changed every 24 h for 3 days.

Viable CFUs
Each well was rinsed twice with 2 ml of phosphate-buffered saline (PBS; Gibco, MD, United States) to remove planktonic cells. The surface of each well was scraped using a cell scraper (Corning, NY, United States), which was then placed in 1 ml PBS, rotated, and shaken for 20 s to homogenize the biofilm bacteria, serially diluted ten times, and plated on TSA agar plates using the drop plate method for a total of three replicates (Herigstad et al., 2001). The control groups were the groups without beads (control 1, C1) and the groups with blank beads (without antibiotics; control 2, C2).
Confocal laser scanning microscopy (CLSM) and fluorescence microscope CLSM and fluorescence microscopy were performed to examine biofilms grown on 35-mm confocal dish plates (Zeiss LSM 980 and ZEISS Axio Imager 2, Germany). The SYTO9/

Statistical analysis
Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, United States). The CFU data were firstly log 10 transformed, and data were expressed as mean ± standard deviation (SD). Student's t-test or one-way analysis of variance (ANOVA) was used to compare differences in CFUs between two or more groups. For one-way ANOVA, post hoc multiple comparisons were conducted using Sidak's test. Statistical significance was set at p ≤ 0.05.

Antibiotic MICs, elution potency, and duration for inhibition of planktonic bacteria
The minimum inhibitory concentrations (MICs) of D, V, and G for ATCC43300 were 0.5, 1, and 32 mg/l, respectively. MRSA was sensitive to both D + G and V + G piggybacking on different materials, with D + G and V + G exhibiting potencies of 31 and 35 days from β-TCP/CS beads and 38 and 40 days from CS beads, respectively. V + G and D + G showed similar potency effects between the two materials, both showing an initial 10-day burst of potency release followed by a flat release until the end of the measurement (Figure 1).

Prevention of biofilm formation and killing efficacy of established biofilms
Bacterial quantification of the colonies in each experimental group was performed using the CFU counting method. In the prophylactic assays (as shown in Figures 2A-D), the number of MRSA biofilms in the control groups [including groups without beads (C1) and groups with blank beads (C2)] was approximately 10 7 to 10 8 CFU/cm 2 at 1 and 3 days. Significant differences were found in the CFUs among the four groups at 1 day, suggesting that CFUs in the D + G and V + G groups were approximately 6 logs less live MRSA colonies than those in the control groups (p < 0.001), either by β-TCP/CS or by CS beads. No significant difference was observed in CFUs between D + G and V + G by post-hoc multiple comparisons, either by β-TCP/CS or CS beads (Figures 2A,B). Similar outcomes were observed 3 days after the respective interventions ( Figures 2C,D).
Regarding the outcomes of the killing assays, after consecutive culturing for 3 days, the number of MRSA colonies in the control groups had grown over 10 8 CFU/cm 2 ( Figures 2E-H). Likewise, significant differences were also identified among the four groups at 1 day, demonstrating that CFUs in the D + G and V + G groups were approximately 4-5 logs fewer live MRSA colonies than those in the control groups (p < 0.001), either by β-TCP/CS or CS beads ( Figures 2E,F). Similar outcomes were also observed after 3 days ( Figures 2G,H). Concerning post-hoc multiple comparisons, results showed that aside from the slightly higher CFUs in V + G compared to D + G at 1 day by β-TCP/CS beads (p < 0.05), no significant differences were noted among other comparisons, either at 3 days by β-TCP/CS beads, or at 1 or 3 days by CS beads.
Concerning potential differences between the two antibiotic carriers, no significant differences were found in CFUs between β-TCP/CS and CS in the prevention experiments, either at 1 or 3 days (Figures 3A,B). For the killing experiments, β-TCP/CS revealed significantly decreased CFUs compared to CS at 1 day, either loaded with D + G or V + G. At day 3, no differences were found between the two materials ( Figures 3C,D). Regarding the outcomes of the single antibiotic groups, as shown in Repeated zones of inhibition (ZOIs) of MRSA, showing the size of the ZOIs (cm 2 ) produced by antibiotic-loaded CS (A) and β-TCP/ CS (B) beads over time. Experiments were performed using three replicates, and data are expressed as mean ± standard deviation (SD).

Frontiers in Microbiology 05 frontiersin.org
Supplementary Figures 1A-D, in the prophylaxis assays, significant differences were found in the CFUs among the four different intervention groups, at 1 or 3 days, either by β-TCP/CS or by CS beads. Post-hoc comparisons showed that the mean CFUs of the G group were significantly higher than those of the D and V groups, at 1 or 3 days, either by β-TCP/CS or by CS (p < 0.001). Similar tendencies were observed in the killing groups ( Supplementary Figures 1E-H).

CLSM and fluorescence microscope
CLSM and fluorescence microscopy were performed to assess the efficacy of different antibiotics, either alone or in combination, for the prevention and eradication of MRSA biofilms. In the prevention assays (as shown in Figure 4A), a visible MRSA biofilm was established in the control group on day 1, with a roughly uniform multilayer structure and a dense accumulation of live and dead bacteria. The majority of the MRSA were live and displayed a green signal. After exposure to antibiotics, an obvious decrease in the number of MRSA colonies was noted in both the D + G and V + G groups without multilayer accumulation. After 3 days of exposure, an increased density of MRSA biofilms was observed in the control group. In contrast, in the antibiotic groups, the number of MRSA colonies remained much lower than that of the control group, although there was a slightly increased density of signals compared to those on day 1 ( Figure 4B).
In the killing assays, all groups were exposed to different interventions after 72 h of mature biofilm formation. As can Methicillin-resistant Staphylococcus aureus (MRSA) CFUs were counted on days 1 and 3 between the prevention and killing groups. Data are expressed as mean ± standard deviation (SD). Prevention groups (A-D); killing groups (E-H); C1, control groups without beads; C2, control groups with blank beads (without antibiotics). ns, not significant; *p < 0.05, ****p < 0.001.
Frontiers in Microbiology 06 frontiersin.org be seen in Figures 5A,B, more dense accumulations of live and dead bacteria were found in the control group on both days 1 and 3, with more dead bacteria interspersed in the multilayer MRSA colonies than in the respective prevention groups. Nonetheless, most of the bacteria remained in the control group. In contrast, dead bacteria were predominant in the antibiotic-treated groups.
In addition, compared with those in the V + G groups, the number of viable bacteria in the D + G groups appeared to be lower on days 1 and 3. As for the single antibiotic groups, the Supplementary Figures 2, 3 showed that compared with those in the control groups, reductions in fluorescence signals were observed in the D, V, and G groups in both prophylactic and killing assays. Similar outcomes were observed in both the β-TCP/CS and CS groups.

Discussion
Despite great advances in medical techniques, IAIs remain the most challenging complication of orthopedic surgery. Bacterial biofilms play a critical role in the occurrence, development, and deterioration of infections and influence treatment efficacy (Stoodley et al., 2011). Thus, effective prevention and eradication of biofilms are of great clinical significance. Radical debridement, followed by local implantation of antibiotics, is the mainstay of IAIs therapy. Therefore, selection of sensitive antibiotics, either alone or in combination, is crucial (Knecht et al., 2018;Aggarwal et al., 2022). Currently, S. aureus is one of the most frequently detected microorganisms causing IAIs, with MRSA accounting for a large percentage. In the past few decades, VAN has always been the first-line antibiotic against MRSA; however, with the increasing incidence of adverse events and increasing reports of resistance and treatment failure, the identification of alternative antibiotics is essential (Micek, 2007). Here, we focused on DAP and compared its efficacy with that of VAN against MRSA biofilms. As VAN is commonly administered in combination with GEN for the clinical management of IAIs, aside from single antibiotics, we also evaluated the efficiency of combinations of antibiotics, including D + G and V + G, for the prevention and eradication of MRSA biofilms.
We found that D + G and V + G shared similar release potency and efficacy against MRSA biofilms, both of which exhibited an effective antibiotic release for 30-40 days, with a rapid release in the first 10 days, followed by a slow and persistent release until the end. Previous studies have also indicated that DAP possesses a release rate similar to that of VAN in PMMA (Hall et al., 2004;Kollef, 2007). In addition, we also noted that antibiotics loaded with β-TCP/CS had a shorter duration of release than those loaded with CS, which may be associated with the fact that β-TCP has a higher porosity, facilitating the release of antibiotics (Zhao et al., 2020). In the prevention assays, the results revealed that β-TCP/CS or CS microspheres carrying D + G and V + G could effectively prevent biofilm formation on days 1 and 3, with significantly fewer CFUs (a reduction of approximately 6 logs). As for the killing experiments, such a reduction reached 4 logs on both days 1 and 3 after implantation of the antibiotics, with similar efficacy between D + G and V + G, demonstrating the possibility of DAP as an alternative antibiotic for local administration against MRSA biofilms.
Although combination antibiotic regimens are frequently administered in clinical practice to widen the antibacterial spectrum, a single administration of such antibiotics should be conducted to better evaluate their efficacy against biofilms. Thus, we assessed the efficacy of single antibiotics D, V, and G in the prevention and eradication of MRSA biofilms. Based on the MIC outcomes, this MRSA strain appeared to be resistant to G, Comparisons of CFUs of MRSA after treatment with β-TCP/CS or CS beads in the prevention (A,B) and killing groups (C,D). Data are expressed as mean ± standard deviation (SD). C2, control groups with blank beads (without antibiotics). *p < 0.05, **p < 0.01.
Frontiers in Microbiology 07 frontiersin.org which still displayed good efficiency in the present study. One underlying factor may be related to the localized presence of high antibiotic concentrations rather than an oral or intravenous scenario, where drug concentrations may be lower and fluctuate due to pharmacokinetics. Our results demonstrated that both D and V may possess better capacities than G to prevent and kill MRSA biofilms, which was supported by fluorography. It should be noted that such different effects may also be caused by higher doses of D (1,000 mg) and V (1,000 mg) than that of G (240 mg) in our study. Growing evidence indicates that DAP possesses fine bactericidal activity against staphylococci in both in vitro and in vivo studies. In an in vitro study (Fuchs et al. 2002), Fuchs et al. analyzed the MICs and minimum bactericidal concentrations (MBCs) of DAP, VAN, linezolid, and quinupristin-dalfopristin against 108 Staphylococcus isolates, and the results revealed that daptomycin was bactericidal against most strains and had the most rapid bactericidal activity. In a recent systematic review and meta-analysis (Samura et al., 2022) Prevention of MRSA biofilm formations on glass surfaces of confocal dish culture plates at days 1 (A) and 3 (B), as observed via CLSM. The biofilms were stained with live and dead stain, where green represents live and red represents dead bacterial cells within the biofilms. The third column in each group is a merge of the previous two columns (live and dead stains).
Frontiers in Microbiology 08 frontiersin.org intermediate-and high-risk bacteremia sources. Although previous studies have indicated the satisfactory efficacy of DAP against Staphylococcus isolates, especially MRSA, most of these were obtained via intravenous administration. Currently, the number of studies focusing on local administration of DAP is limited. Several reports have analyzed the efficacy of DAP loaded with PMMA, and the outcomes showed its good antimicrobial capacity without altering the structural characteristics in terms of its release potency (Rosslenbroich et al., 2012;Tan et al., 2012;Ferreira et al., 2015). While our study found that aside from PMMA, DAP can be loaded by β-TCP/CS and CS, and both showed good outcomes in the prevention and eradication of MRSA biofilms. In addition to its antimicrobial capacity, DAP also has advantages such as lower mortality and relapse rates, better tolerability, and fewer adverse events (Lamp et al., 2007;Moenster et al., 2012;Moise et al., 2016;Schweizer et al., 2021). Although DAP has advantages, its wide application remains limited because of its high cost and the uncertainty regarding the dose for pediatric patients under 12 years (Heidary et al., 2018). Our study has some limitations. First, our experiment was conducted using only in the vitro test setup. In vivo factors, such as blood flow, movement, protein binding, and involvement of immune cells, may lead to alterations in antibiotic activity. In addition, the model we used in the present study was initially developed for catheter-related blood infections that often grow biofilms at the solid-liquid interface (bottom of the microwell) or the solid-liquid-air interface (wall of the microwell), whereas biofilms in orthopedic IAIs are formed at the device-tissue interface. Thus, the current model may not fully reflect the status of biofilms during IAIs. Second, in the simulated biofilm prevention and killing experiments, the tests were only conducted on days 1 and 3, which is insufficient, and a longer culture time is warranted to better evaluate the efficacy of different antibiotics. Third, we selected only the standard MRSA strain ATCC43300 for the experiment. Different clinical MRSA strains should be used to comprehensively assess the efficacy of DAP against MRSA. Finally, the concentrations (mix ratios) of antibiotics with β-TCP/CS or CS in our study were set, with respect to clinical use, and the optimal DAP concentration remains uncertain. Therefore, subsequent in vitro and in vivo experiments should be performed to evaluate the efficacy and safety of different concentrations of local DAP for the management of IAIs. Despite these limitations, Killing of biofilm formed by MRSA on glass surfaces of confocal disc culture plates on days 1 (A) and 3 (B) after biofilm formation, as observed via CLSM. Biofilms were stained with live and dead stains; green, live bacterial cells within biofilms; red, dead bacterial cells within biofilms. The third column in each group is a merge of the previous two columns (live and dead stains).
our study is valuable, as it provides preliminary evidence regarding the local administration of DAP against MRSA biofilms, providing an alternative selection of antibiotics for the treatment of IAIs.
Our experiments demonstrated that D + G loaded with β-TCP/CS and CS displayed similar efficacy to V + G in preventing the formation of, as well as killing, MRSA biofilms. Although VAN remains the first-line agent for the treatment of IAIs, DAP may be a suitable and effective alternative when VAN resistance develops or patients cannot tolerate VAN.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

Author contributions
NJ and BY: conceptualization, writing-review and editing, supervision, project administration, and funding acquisition. XZ, NJ, and BY: methodology. XZ, PC, and BY: validation. XZ, H-yW, and NJ: formal analysis. XZ, PC, H-yW, R-jZ, and M-rS: investigation. YZ and R-jZ: data curation. XZ, PC, and NJ: writing-original draft preparation. All authors contributed to the article and approved the submitted version.

Funding
This study was funded by the National Natural Science Foundation of China (Grant Nos. 81830079, 82172197 and 82272517).


Hair Sample Analysis as a Method of Monitoring Exposure to Bisphenol A in Dogs

Bisphenol A (BPA) is an organic substance widely used in the plastics industry. It penetrates food and environment and, as an endocrine disruptor, has detrimental effects on human organisms. Pet animals, which live in the immediate vicinity of humans, are also exposed to BPA; however, knowledge regarding the exposure of dogs to this substance is extremely scarce. This is the first study in which hair analysis has been used to biomonitor BPA in 30 dogs using liquid chromatography and tandem mass spectrometry techniques. The presence of BPA in concentration levels above the method detection limit (1.25 ng/g) was noted in 93.33% of samples. BPA concentration levels were found to range from 7.05 ng/g to 436 ng/g (mean 81.30 ng/g). Statistically significant differences in BPA concentration levels were found between animals with physiological weight and animals with abnormal weight (skinny and obese). In turn, differences between males and females, as well as between young, middle-aged and old dogs, were not statistically significant. The obtained results have clearly shown that hair analysis is a useful method to evaluate the exposure of dogs to BPA. This study also confirmed that dogs are exposed to BPA to a large extent, and this substance may play a role as a pathological factor in this animal species. However, many aspects connected to the influence of BPA on canine health status are unclear and need further study.

Introduction
Bisphenol A (BPA) (chemical name: 4,4 -(Propane-2,2-diyl)diphenol) is an organic synthetic substance produced during the condensation of acetone and phenol [1,2]. Since products containing BPA are resistant to destruction and light, as well as relatively inexpensive, this substance is commonly used in the plastics industry [2]. BPA is contained in various objects of everyday use, including, among others, bottles, food containers, electronic equipment, household goods, furniture, and even dental fillings [2,3].
It is known that BPA can penetrate plastic, food, soil, and air, as well as drinking, tap, and surface water [4][5][6][7]. Due to the common use of BPA in industry, the presence of this substance has been found in the environment all over the world; it has even been detected in Antarctic ice [8].
BPA also penetrates living organisms through the digestive tract, respiratory system, and skin [2,[9][10][11]. Moreover, it is known that prenatal exposure to BPA is also possible because this substance can cross the placenta [12]. Previous publications have described the presence of BPA in human blood, urine, and milk, as well as in domestic and wild animals [13][14][15][16].

Hair Sample Collection
Hair samples were collected from 30 clinically healthy male and female dogs of various breed, age and body condition score (BCS). They were selected randomly from animals whose owners responded to the study announcement, consented to the hair sampling, and provided some information about their animals. According to the information obtained from the owners, all dogs included in the study were indoor dogs living in Olsztyn-a city of about 170,000 inhabitants located in north-eastern Poland-who went out to walk with their owners several (from four to seven) times a day. All dogs included in the study had a similar diet that included mainly canned and dry commercial canine food, and food and water were served in plastic bowls. The number of dogs included in the study was limited by the number of dog owners interested in the study willing to provide a small amount of information regarding their dog.
Since hair sampling is completely non-invasive and the collection of samples was conducted during care treatments, the present study did not require the consent of the Ethical Committee. This is consistent with the Act for the Protection of Animals for Scientific or Educational Purposes of 15 January 2015 (Official Gazette 2015, No. 266), applicable in the Republic of Poland. The exact data concerning dogs included in the experiment are shown in the Supplementary Materials, Table S1.
Hair samples (about 2 g each) were collected from the same place from all dogs. Namely, hair was cut from the abdomen as close to the skin as possible. Immediately upon collection, hair samples were wrapped in aluminium foil and stored in the dark at room temperature until further study. During storage, the hair samples did not contact materials that could contaminate them with BPA.
Before further examination, hair samples were washed to remove surface contaminations. The hair was first washed in ultrapure water, followed by SDS (0.1%, w/v), and then twice with ultrapure water. In each washing step, the sonication of hair samples for 5 min was made. After washing, the hair was cut into pieces of 2-3 mm long, dried, wrapped in aluminium foil, and stored in the dark at room temperature until further analysis. Three samples of the hair from each dog were subjected to analysis.

BPA Extraction and Analysis of Its Levels
Analysis was performed following the method previously described by Martín et al. [35,38] with slight modifications. Washed hair samples (100 mg) containing BPA-d 14 (12.5 ng) were incubated with a mixture of methanol and HAc (2 mL, 85:15, v/v) in 10 mL glass centrifuge tubes. Incubation lasted for 12 h at 38 • C. The samples were then cooled to room temperature, extracted with 3 mL of acetone in an ultrasonic bath for 15 min, and centrifuged for 10 min at 2900× g. The liquid phase was separated into a clean tube and evaporated to dryness under a nitrogen stream at room temperature. The residue was reconstituted in 0.25 mL of methanol, filtered through a 0.22 µm nylon filter. An aliquot (10 µL) of the extract was injected into the instrument.
Liquid chromatography-tandem mass spectrometry analysis was made with an Agilent 1260 Infinity II (Agilent, Santa Clara, CA, USA) and a HALO C-18 Rapid Resolution (50 × 4.6 mm i.d., 2.7 µm particle size) column. The mobile phase was composed of a 10 mM ammonium acetate solution (solvent A) and MeOH (solvent B). The elution gradient was as follows: for 0-14 min, the linear gradient of solvent B rose from 28 to 70% at a flow rate of 0.6 mL/min; this rose from 70% to 80% after 5 min, increased to 100% at 6 min, and then held for 2 min. The column temperature was maintained at 30 • C.
The mass spectrometer was operated in negative electrospray ionisation mode. [M-H] − was used as a precursor ion (m/z 227). The analyses were performed in multiple reaction monitoring (MRM) mode. Two transitions were selected (m/z 227 → 133 for quantification and m/z 227 → 211.8 for confirmation purposes). The other MS parameters were as follows: fragmentor-126 V; collision energy-24 eV; capillary voltage-3000 V; drying gas flow rate-9 L/min; drying gas temperature-350 • C; nebulizer pressure-40 psi. Figure S1 shows the BPA mass spectrum.

Background and Quality Control
An important aspect related to BPA analysis is blank contamination owing to the widespread use of plastics and epoxy resins in laboratory materials. As far as possible, plastic materials were avoided in preparing the samples. Moreover, to remove any possible BPA contamination, all glassware were washed with milli-Q water and sonicated with acetonitrile. The material was then rinsed with Milli-Q water and dried in an oven at 120 • C for 4 h.
A quality assurance/quality control protocol was established to guarantee credible and precise results. The protocol contained the use of spiked samples (50 ng/g), BPA standard (20 ng/mL), procedural blanks, and methanol injections into each batch of samples (15). Procedural blanks were processed in the same manner as the hair samples. An average concentration below the method quantification limit (MQL) of BPA was achieved in all analysed procedural blanks. The BPA concentrations in hair samples were calculated by matrix-matched calibration curves. The validation parameters are summarized in the Supplementary Materials, Table S2. The method detection limit (MDL) and MQL were 1.25 ng/g and 4.2 ng/g, respectively. In the absence of certified reference materials, recovery assays with spiked samples using matrix-matched calibration were used to validate the method. Recovery rates ranged from 94% to 99%, while inter-and intra-day variability was under 6%. Figure S2 shows the MRM chromatograms obtained for spiked samples with BPA at low, medium, and high concentration levels and Figure S3 shows the MRM chromatogram of a real sample analyzed.

Statistical Analysis
The statistical analysis was performed using GraphPad Prism version 9.2.0 (GraphPad Software, San Diego, CA USA). In the case of a comparison between two groups (male dogs versus female dogs), a non-parametric Mann-Whitney test was used, and in the case of a comparison between three groups (thinner dogs versus dogs with normal weight versus dogs with obesity, and young dogs versus middle-aged dogs versus old dogs) a Kruskal-Wallis test was used. The differences were considered statistically significant at p < 0.05.

Results
In this study, BPA concentration levels were evaluated in 28 out of the 30 dogs included in the experiment (93.33% of analysed samples); in two dogs, the BPA levels were below the method detection limit (MDL). The concentration levels of BPA were different in particular animals and fluctuated from 7.05 ng/g to 436 ng/g with an arithmetic mean of 81.30 ng/g. The data obtained during the present study are summarised in Table 1.
During the present study, the mean concentration levels (±SD) of BPA in male dogs amounted to 58.16 ± 37.01 ng/g, and were slightly lower than those observed in females, which achieved values of 90.56 ± 120.1 ng/g. In the male group, the highest BPA concentration level amounted to 126 ± 1.65 ng/g and was noted in a Poodle, and in the female group it was 436 ± 7.16 ng/g (observed in a Golden Retriever) (Table 1). Nevertheless, there were no intragender statistically significant differences in BPA concentration levels ( Figure 1). Some differences in BPA concentration levels were also noted depending on the age of the animals. In young animals, the mean BPA concentration levels amounted to 60.47 ± 41.78 ng/g and increased with age to 82.98 ± 115.3 ng/g and 108.90 ± 146.7 ng/g in middle-aged and old animals, respectively. In young dogs, the highest concentration level of BPA amounted to 126 ± 1.65 (noted in a Poodle), in middle-aged dogs this amounted 436 ± 7.16 ng/g (noted in a Golden Retriever), and in old dogs this amounted to 307 ± 9.56 ng/g (noted in a Poodle) (Table 1). However, these differences were not statistically significant (Figure 2). Some differences in BPA concentration levels were also noted depending on the age of the animals. In young animals, the mean BPA concentration levels amounted to 60.47 ± 41.78 ng/g and increased with age to 82.98 ± 115.3 ng/g and 108.90 ± 146.7 ng/g in middleaged and old animals, respectively. In young dogs, the highest concentration level of BPA amounted to 126 ± 1.65 (noted in a Poodle), in middle-aged dogs this amounted 436 ± 7.16 ng/g (noted in a Golden Retriever), and in old dogs this amounted to 307 ± 9.56 ng/g (noted in a Poodle) (Table 1). However, these differences were not statistically significant ( Figure  2).   Some differences in BPA concentration levels were also noted depending on the age of the animals. In young animals, the mean BPA concentration levels amounted to 60.47 ± 41.78 ng/g and increased with age to 82.98 ± 115.3 ng/g and 108.90 ± 146.7 ng/g in middleaged and old animals, respectively. In young dogs, the highest concentration level of BPA amounted to 126 ± 1.65 (noted in a Poodle), in middle-aged dogs this amounted 436 ± 7.16 ng/g (noted in a Golden Retriever), and in old dogs this amounted to 307 ± 9.56 ng/g (noted in a Poodle) (Table 1). However, these differences were not statistically significant ( Figure  2).  Differences in BPA concentration levels were also noted depending on the weight of the animals. The lowest mean BPA concentration levels, amounting to 26.62 ±20.35 ng/g, were observed in dogs with physiological weight. This value was statistically significantly lower than the mean BPA concentration level noted in thinner dogs (91.08 ± 28.55 ng/g) and dogs with obesity (124.6 ± 137 ng/g) (Figure 3). The highest BPA concentration levels in skinnier dogs amounted to 126 ± 1.65 ng/g (noted in a Poodle), in dogs with physiological weight, 40.1 ± 2.19 ng/g (noted in a mongrel), and in dogs with obesity, 436 ± 7.16 (noted in a Golden Retriever) ( Table 1).
were observed in dogs with physiological weight. This value was statistically significantly lower than the mean BPA concentration level noted in thinner dogs (91.08 ± 28.55 ng/g) and dogs with obesity (124.6 ± 137 ng/g) (Figure 3). The highest BPA concentration levels in skinnier dogs amounted to 126 ± 1.65 ng/g (noted in a Poodle), in dogs with physiological weight, 40.1 ± 2.19 ng/g (noted in a mongrel), and in dogs with obesity, 436 ± 7.16 (noted in a Golden Retriever) ( Table 1).

Discussion
The results obtained in this study have shown that dogs are widely exposed to this substance. This is in agreement with previous observations, which have described the presence of BPA in the serum and urine of pet animals ( Table 2).

Discussion
The results obtained in this study have shown that dogs are widely exposed to this substance. This is in agreement with previous observations, which have described the presence of BPA in the serum and urine of pet animals ( Table 2). Dogs and cats live in immediate proximity to their owners in the same environment, as a result of which humans and pet animals are exposed to the same environmental pollutants. In animals, just like in humans, one of the main sources of BPA penetrating the body are food and drinking water. It is commonly known that BPA is present in various food products around the world, including fish, meat, milk, and food of plant origin, and the BPA concentration levels clearly depend on the place where the studies have been performed [3,4,42,43]. Moreover, BPA has also been noted in canned food for cats and dogs. In cat food, BPA concentration levels fluctuate from 13 to 136 ng/g [30], and in dog food, depending on the study, from 11 to 206 ng/g [29,30]. In turn, the main cause of BPA presence in canned pet animal food is not the contamination of raw materials used for food production, but the penetration of BPA from the inner layers of cans [29].
The second important source of exposure to BPA may be connected to polluted indoor air and house dust. Previous studies conducted in various world regions have reported that concentration levels of BPA in house dust vary greatly among countries and reach from 9.6 up to 32,000 ng/g [44][45][46]. BPA is also present in indoor air, and its concentration levels depend on the part of the world and degree of industrialisation [47]. The high impact of BPA in indoor dust and air on the degree of pet animal exposure to this substance is confirmed by the fact that the blood serum of indoor cats has been found to contain higher BPA concentration levels than cats with outdoor access [15].
Animal toys, training devices, and bowls may also influence pet animal exposure to BPA. These items are often made from plastic materials containing BPA because in the production of items intended for animals (contrary to items for people) BPA-free plastics are usually not used. However, it is known that BPA may penetrate from canine toys and training devices to the saliva during chewing [48] and be absorbed in the intestine and through the mucosal layer of the mouth [49]. Humans are exposed to BPA in numerous situations which are irrelevant to animals, for example, through contact with thermal paper, electronic equipment, beverages in plastic bottles, and dental fillings [2,50,51]. For these reasons, animals may be less exposed to BPA than humans. However, because exposure to BPA is extremely varied in different parts of the world, this cannot be stated unequivocally. Moreover, many issues are not clear, for example, why BPA concentration levels were much higher in cats than in dogs when samples were taken in the same area [28]. This probably results from (so far unknown) intraspecies differences in BPA metabolism. However, comparing the results from one country, we can see that BPA concentration levels in dogs are lower than in humans. For example, in Poland, concentration levels of BPA in human hair were found to range from 26.1 to 1498.6 ng/g [52], while in dogs they ranged from < MDL to 436 ng/g (this work). However, it should be remembered that studies have been performed in various environments (various cities), which may also affect the obtained results.
A comparison of the results obtained in the present study with previous observations conducted on dogs or cats in other countries (Table 2) is also very difficult since, until now, canine or feline hair samples have not been analysed for BPA content. Previous studies have shown that mean BPA concentration levels in urine and serum did not exceed 23 ng/mL and 3 ng/mL in cats and dogs, respectively (Table 2), and were much lower than those obtained in the present study. This fact may result not only from differences in exposure to BPA and various environmental factors influencing this exposure, but also from extremely varied matrices. It is known that BPA in urine is extracted daily, and BPA in serum is transported to various tissues and urine, while it is unknown exactly how it may accumulate in the hair. Moreover, the analysis of hair takes into account two sources of BPA: (a) external, in which BPA penetrates directly from the environment into the hair; and (b) internal, in which BPA gets inside the hair through the blood and hair follicle/root. These facts may cause BPA concentration levels noted in canine hair to be higher than those noted in urine or blood serum.
During the present study, statistically significantly higher BPA concentration levels were noted in obese and thinner dogs than dogs with physiological weight. Previous studies have reported that BPA is an important obesogenic factor in humans [53,54]. This is connected with BPA influence on adipose tissue and changes in the production and secretion of adipokines, which take part in the regulation of appetite and satiety centres, and are therefore important factors in the pathogenesis of obesity [55,56]. The results obtained in the present study suggest similar BPA activity in dogs, contrary to cats, for whom statistically significant differences in BPA concentration levels between obese animals and animals with physiological weight have not been described [15]. In turn, the reason for the higher BPA concentration levels noted in the present study in thinner dogs is not clear, and may be connected with endocrinal activity disturbances related to insufficient weight.
During the present study, it was noted that BPA concentration levels slightly increased with the age of the animals, but differences between age groups were not statistically sig-nificant. In terms of the association between BPA concentration levels and age in humans, the issue is not completely clear. Some studies have shown higher BPA concentration levels in children than adults [39,57], which may be explained by the fact that children are more exposed to BPA in relation to their weight than adults. Moreover, it suggests that metabolic pathways that are not fully developed might play a part in BPA transformation. However, other studies have shown higher BPA concentration levels in adults in comparison with children [35]. Higher concentration levels of BPA in older individuals have also been noted in cats [15], which may be explained by the fact that gastrointestinal disturbances (and therefore changes in absorption and the metabolism of BPA) occur more often in older individuals.
An explanation of the correlation between BPA concentration levels and age is difficult. Although the exact mechanisms of BPA metabolism in particular mammal species are not clear, various factors, including diet, lifestyle, industrialisation, and-in the case of humans-dental fillings or workplace location, may modulate exposure to BPA and therefore the concentration levels of this substance in an organism [2][3][4].
A similar situation concerns intragender differences in terms of concentration levels of BPA. During the present study, statistically significant differences in BPA concentration levels between male and female dogs were not observed, although the value was slightly higher in females. Previous studies conducted on humans have described various observations. Some of them have shown higher BPA concentration levels in men, which, according to the authors, is related to higher androgen levels [58], while others have reported higher concentration levels of this substance in girls than in boys [39]. Other studies, including studies performed on men and women living in the same household, collected on the same day, have not shown intragender differences in BPA concentration levels [59]. Moreover, some studies have reported that, although the levels of total BPA in men and women are similar, intragender differences in levels of BPA metabolites have been found [60]. Intragender differences in BPA concentration levels may be connected to various metabolism rates and hormonal activity in males and females, but also (as in the case of age-dependent differences in BPA levels) with other various factors (for example, diet or dwelling place).
An interesting issue is also the correlation between the degree of exposure to BPA and the breed of the dog. Due to the fact that, in the present study, dogs of various breeds (from one to several individuals of the same breed) were examined, the present results do not clearly answer the question of whether such correlations exist. Clear differences in BPA concentration levels between particular animals within the same breed (for example, in the case of a Schnauzer, where values fluctuated from 7.75 ± 0.65 ng/g to 178 ± 7.40 ng/g) may suggest that exposure to BPA is not correlated with the breed, but with other environmental factors. However, further studies on this issue are necessary, and should involve larger groups of dogs of the same breed.
It should be noted that the analysis of hair for the biomonitoring of BPA concentration levels is a relatively new method, which until now has only been used in a few studies (Table 3). MDL-method detection limit; LOQ-limit of quantification.
Both previous studies and the present work have clearly shown that hair sample analysis may be a good alternative to studies using "classic" matrices, such as urine or blood serum, due to its non-invasive character and easiness of sample collection. However, despite its advantages, hair analysis has some limitations. As mentioned above, since studies on hair and hair samples take into account external and internal exposure to BPA, in this method the separation of externally deposited from internally deposited BPA is, therefore, practically impossible. Moreover, hair analysis is not the correct method for studies on short-term fluctuations in BPA concentration levels under rapidly changing factors, because this substance accumulates in the hair over a long time. Therefore, hair analysis is adequate for the monitoring of long-term exposure to BPA, and urine analysis seems to be the best method to study short-term changes.
In spite of these limitations, the results obtained in the present study show that hair analysis may be used in explaining the many aspects associated with the exposure of dogs to BPA. The easy and stress-free collection of hair samples will enable comprehensive studies on large experimental groups concerning the correlation between the degree of exposure to BPA and risk of various pathological states in dogs, e.g., liver and gastrointestinal diseases, diabetes, obesity, endocrinal disturbances, and neoplastic processes. Although information about BPA-induced pathological processes in dogs is rather scarce, previous studies on experimental animals and epidemiological reports on humans [4,[17][18][19][20][21][22][23] strongly suggest that BPA may influence both the above-mentioned diseases and other disturbances in dogs. The long-term biomonitoring of dogs' exposure to BPA using hair sample analysis may also be used in dog breeding, as it may eventually elucidate the influence of BPA on disturbances to the reproductive processes in various canine races. A correlation between the degree of exposure to BPA and the functionality of the canine reproductive system is very likely, because BPA-induced disturbances in reproductive processes in other species are relatively well-known [2,4]. Another problem which may be explained with hair samples analysis is the exposure of dogs to BPA through food, toys, and other equipment involved in dog keeping (such as bowls, collars, leashes). Previous studies strongly suggest that food and/or toys may be a source of BPA that penetrates canine organisms [29,30,48,49]. Moreover, it should be pointed out that this problem concerns not only pet dogs, but also animals working in security services in public and military areas. Therefore, the biomonitoring of BPA in dogs is an important problem in modern veterinary and dog breeding, and the analysis of hair samples (due to easy and stress-free collection) may be one of the methods which will help to solve this problem. Of course, due to very limited previous reports, this issue requires further comprehensive research.

Conclusions
To conclude, the present study, which may be the first step to further investigations on BPA in pet animal hair, has clearly shown that hair analysis may be useful in determining the degree of exposure of dogs to BPA. The obtained results have also confirmed that dogs are highly exposed to BPA and, hence, this substance may be important as a pathological factor in this species. However, the exact explanation of all aspects connected to BPA influence on the health status of dogs needs further comprehensive and multidirectional study. This might involve evaluating BPA metabolism in dogs, BPA-induced disturbances in internal organ functionality and canine diseases, or the environmental factors affecting the degree of exposure to BPA in dogs.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijerph19084600/s1, Table S1: Characterization of dogs included into the study; Table S2: Linear range, MDLs, MQLs, recovery and precision of BPA in fur samples; Figure S1: Mass spectrum of BPA; Figure S2: MRM Chromatograms of fur samples spiked at low, medium and high concentration levels; Figure S3: MRM Chromatograms of a real fur sample and a blank matrix.