Overview of Mink Immunity and Resistance to Pseudomonas aeruginosa

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Introduction
Pseudomonas aeruginosa (P. aeruginosa) is one of the most host-susceptible pathogenic bacteria that causes a wide range of acute and chronic infections in humans and animals [1,2]. In minks, P. aeruginosa induces serious pulmonary diseases to deaths and subsequent economic losses, as an environmentally ubiquitous, extracellular, and opportunistic pathogen [3][4][5]. For example, it can cause fatal pathogenic infectious hemorrhagic pneumonia with cystic fbrosis, chronic bronchitis, and bronchiectasis, when the host resistances decrease [6][7][8][9]. In the real farming environment, the interactions of P. aeruginosa and host immunity and resistance determine the pathogenic mechanism, so it can survive in the varied environments based on the multiple bacterial virulence factors and genetic fexibility [6,7].
Many previous mink studies have worked on the pathology, pathogenesis, serology, antimicrobial resistance, virulence gene, and related diseases of P. aeruginosa to understand its molecular infectious mechanism, to fnd the clinically suitable antibiotics, and to provide the best strategic treatment solutions [10][11][12][13][14][15]. In the meantime, mink vaccinations against P. aeruginosa were developed for clinical treatments, such as bacterin, multicomponent (common protective antigen of P. aeruginosa mixed with toxoids of protease and elastase of the bacillus), and other technological vaccines to protect minks with efective immunity from hemorrhagic pneumonia [5,16,17].
However, the relationship study of P. aeruginosa infections with mink immunity and resistance is relatively less so far and it is still required to be further studied and deeply elaborated. Here, we summarized the characterizations and infectious mechanisms of P. aeruginosa and the mink immunity and resistance to P. aeruginosa from the previous studies to understand the relationships of P. aeruginosa infections with the host immunity that could provide the valuable understandings of pathogenesis of mink hemorrhagic pneumonia for further related research and to contribute to the potential clinical treatments of P. aeruginosa infections and to avoid the fatal hemorrhagic pneumonia spreading in the future.

Characterization of Pseudomonas aeruginosa
Several previous P. aeruginosa studies have characterized the isolates for strain type, prevalence, occurrence rate, antibiogram, and molecular pattern [18,19], where they found the following: (1) the respiratory tract was the most common place for P. aeruginosa multiplication; (2) the signifcant declines of β-lactam and aminoglycoside susceptibility rates were only found in Europe regions; (3) β-lactamase production and multiple drug resistance efux pumps contributed to the presence of antibiotic-resistant P. aeruginosa isolates [18,19]. Tose characterization studies of P. aeruginosa aim to know the resistance rates of tested antibiotics among diferent strains, to understand a variety of resistance mechanisms through diferent phenotypes, to characterize the transcriptional regulator of catabolic genes (phhA, HPD, hmgA, dhcA) that were induced during P. aeruginosa growth in cystic fbrosis (CF) sputum, to demonstrate the virulence factors involved in adherence and secretion systems, and to reveal the potential phage therapy candidate for the infection treatment [20][21][22][23][24].
2.1. Pseudomonas aeruginosa. P. aeruginosa is a heterotrophic, motile, Gram-negative rod-shaped bacterium belonging to the class of gamma-Proteobacteria in the family Pseudomonadaceae with around 1-5 µm long and around 0.5-1 µm wide [25]. Based on diferences in the O-antigen of lipopolysaccharide, Liu and Wang identifed 20 diferent serotypes of P. aeruginosa [26]. P. aeruginosa owns a large size of genome that is estimated to 6.4 Mb on average refecting the extreme versatility [27]. P. aeruginosa strains are normally subdivided into two major groups that are group I including strain PAO1 and group II including strain PA14 [25,28]. In the clinical applications, the acquisition of resistance genes via horizontal gene transfer and the mutations in genes can cause antimicrobial resistance of P. aeruginosa to up-regulation of efux pumps, β-lactamase or changes in porins [25]. P. aeruginosa can be easily found in almost any human and animal environments, even if it can be hardly isolated from a number of environments including soil and water. Due to the tendentious formation of multicellular bioflms with a wide variety of antibiotic resistance mechanisms, it majorly caused humans illness and death under immunosuppressive and chronic conditions, so such infections are difcult to treat in these patients [25].

Virulence Factors of Pseudomonas aeruginosa.
Lipopolysaccharide (LPS) normally contains a tripartite structure that are lipid A, core oligosaccharide, and O antigen polysaccharide, where lipid A is the hydrophobic moiety that anchors LPS to outer leafet together with core oligosaccharide to maintain the integrity of the outer membrane. O antigen polysaccharide is connected to the core with a polymer made of repeating oligosaccharide units in direct contact with the external milieu [29]. Te complete LPS with tripartite structure is normally called "smooth," but LPS molecules referred to lipooligosaccharides are normally called "rough" only with the lipid A and core [30]. Particularly, the low outer membrane permeability of P. aeruginosa contributes its high intrinsic resistance to antiseptics and antibiotics because the general outer membrane porin (OprF) displays structural, adhesion, and signaling functions [31]. Fito-Boncompte et al. revealed the involvement of OprF in P. aeruginosa virulence through modulation of the quorum-sensing network, so OprF is required for P. aeruginosa virulence. In addition, the absence of OprF results in secretion of ExoTand ExoS toxins through the type III secretion system (T3SS), impaired adhesion to animal cells, and production of the quorum-sensingdependent virulence factors pyocyanin, elastase, lectin PA-1L, and exotoxin [32]. Recently, Remans et al. [33] indicated that the number of lipoproteins in P. aeruginosa was 175 reduced from 185 compared with the previous studies; lipoproteins are translocated from the cytoplasm, and their Nterminal signal peptide is cleaved by the signal peptidase II [33,34].
As a bioflm research model that enables drug resistance with bioflm formations to cause chronic infections, P. aeruginosa can produce such robust bioflms in immunocompromised patients with severe problems; furthermore, the unique bioflm properties complicate eradication of bioflm infection that resulted in the chronic infection developments [35,36]. Pseudomonads produce several bioflm matrix molecules (polysaccharide, nucleic acid, and protein) and accessory matrix components to aid bioflm formation and adaptability under varying conditions [37]. Tielen et al. found the infuences of secreted enzymes EstA, LasB, and LipC on formation and architecture of mucoid P. aeruginosa bioflms because of changes in EPS composition and properties and their cell motilities [38].

Pseudomonas aeruginosa Infections
P. aeruginosa can cause various host infections under immunocompetent and immunocompromised conditions such as folliculitis, osteomyelitis, otitis externa, and pneumonia. Meanwhile, its extreme versatility together with a wide array of antibiotic resistances and dynamic defenses make it extremely challenging to be treated [39][40][41]. Te pathogenicity and infection symptoms of P. aeruginosa in human, mink, and other species are listed in Table 1.

Mink Infections of Pseudomonas aeruginosa.
Due to P. aeruginosa infections, minks develop acute, contagious, and fatal hemorrhagic pneumonia as the only known animal species in healthy individuals (Table 1). In Denmark, hemorrhagic pneumonia in minks was frstly reported in 1953 that caused variable mortalities ranging from 1% to 75% [4,5,10,11]. In China, P. aeruginosa pneumonia in minks was frstly reported in 1985 with percentages of mortality ranging from 6% to 54%. Te outbreaks appear Human Chronic obstructive pulmonary disease (COPD) Breathing difculty, coughing, wheezing persistently, and producing sputum [8,43] Human Cystic fbrosis (CF) Cough persistently, produce thick sputum, wheeze, exercise intolerance, repeated lung infection, infamed nasal passages, and recurrent sinusitis [7,48] Mink Hemorrhagic pneumonia Lung infammatory consolidation and lung bleeding [4,5] Chicken Pericarditis Cough, fever, and heart palpitations [44] Horse Endometritis Purulent vaginal discharge and an excess of echogenic fuid within the uterine lumen [45] Pet Otitis externa Head shaking, ear pain and swelling, and ceruminous gland infammation [46,47] Veterinary Medicine International every year in diferent farms, particularly from August to early December, when all-age minks are afected with typical symptoms, such as nose bleeding, mouth bleeding, pulmonary hemorrhage, and pleural efusions [3,42]. Qi et al. revealed the genetic similarities and antimicrobial susceptibility profles of P. aeruginosa from clinical cases of mink hemorrhagic pneumonia that facilitated prevention and control of such disease in China [3]. Bai et al. [42] proved the genetic diversity of mink P. aeruginosa isolates from different farms and indicated the potential risk to human health, especially for patients with CF as a result of the unique P. aeruginosa strains that was acquired in the environment of the patient.

Human and Other Species Infections of Pseudomonas aeruginosa.
In humans and other species, P. aeruginosa is known as an opportunistic pathogen that enables establishment of an infection when the host is immune compromised or susceptible in other ways [1], so it can cause pulmonary disease, pericarditis, endometritis, otitis externa, etc. (Table 1). P. aeruginosa isolated from adult sputum cultures with chronic obstructive pulmonary disease (COPD) displayed two distinct patterns of carriage: short-term colonization followed by clearance and long-term persistence. Diferent manifestations of P. aeruginosa were observed in COPD that causes acute exacerbations and probably chronic infection in a subset of adults [43]. Generally, airways of patients with CF can be infected by diferent bacterial species, but P. aeruginosa infection causes the greatest burden of morbidity and mortality, which begins with a period of recurrent, intermittent colonization from the environment or from a protected niche within the patient. Such chronic infections of the CF airway provide a valuable opportunity to study bacterial evolution in a complex natural environment [7].
Hassan et al. [44] performed a study to probe the antimicrobial resistance and virulence gene profling of P. aeruginosa in broiler chickens with pericarditis. Tey found that P. aeruginosa was highly virulent and resisted most of the therapeutic agents that bore hazards for poultry industry and represented a public health concern [44]. Kidd et al. studied the relatedness among P. aeruginosa isolates to suggest that most equine genital P. aeruginosa infections were probably acquired from mechanisms other than direct horse to horse [45]. Haenni et al. found the drug resistance to P. aeruginosa with a low proportion of diseased cows and horses, but resistance phenotypes were more frequently observed in dogs. Terefore, the monitorization is important as a result of the animal-to-human transfer between pets and humans, especially for patients with CF [46]. Multidrug efux pumps are essential for inhibitor development to P. aeruginosa treatment, so Poonsuk and Chuanchuen concluded that the MexXY multidrug efux pump could act aminoglycoside resistance to P. aeruginosa infection in dogs and cats, which indicates the existence of uncharacterized aminoglycoside-resistance mechanisms [47].

Immunity and Resistance to
Pseudomonas aeruginosa Sindeldecker and Stoodley [49] summarized the various antibiotic resistance and tolerance mechanisms of P. aeruginosa including classic mutation driven resistance, adaptive resistance, persister cells, small colony variants, phoenix colonies, and bioflms. Tey evaluated the antibiotic surviving isolates to combat the rising number of recurrent and recalcitrant infections by characterizing these phenotypes after distinguishing various phenotypes [49].

Immunity and Resistance to Pseudomonas aeruginosa with
Hormones. Clearly, various hormones can afect immune defense, such as prolactin and melatonin, because immune and endocrine systems communicate each other to infuence each other [50][51][52]. At the onset of fall, hormones fuctuations could possibly cause the hemorrhagic pneumonia in minks because the hormones involved in change of fur and adaptation to altered photoperiod exhibit fuctuations, such as a slow rise in testosterone, a rise in melatonin, and a fall in circulating prolactin [53,54]. Te catecholamines promoted in vitro P. aeruginosa growth, while corticosteroids and catecholamines were also believed to be important as suppressors and modulators in the immune system [55][56][57][58][59][60]. Corticosteroids were commonly used combined with antimicrobials to treat infectious diseases for infammatory process control and potential toxicity minimum of antimicrobials to avoid sequelae [61,62]. Rodrigues et al. provided in vitro and in vivo efects of antimicrobials and glucocorticoids combinations that were the interference evidences of dexamethasone on the pharmacological activity of clinically antimicrobial drugs against bioflms and planktonic cells of P. aeruginosa [61]. In addition, Satoh et al. [62] established fatal pneumonia with bacteraemia in mice after dexamethasone treatment using intratracheal infection of P. aeruginosa to elucidate in vivo mechanisms in the pulmonary defense impairment. Tey found the suppressed production of tumour necrosis factor alpha (TNF-α) during the early phase of pneumonia under dexamethasone treatment, so such TNF-α producing inhibition in the lung could be responsible for the progression of the fatal pneumonia [62].
Melatonin could play an important role as well in regulation of immune defense by stimulating acute infammation and attenuating chronic infammation [50], where females were considered to show a stronger immune response compared to males that was probably caused by the onset of production of testosterone for the apparent increased susceptibility of male minks that were infected with P. aeruginosa [63][64][65]. Melatonin has also showed antibacterial efects by reducing intracellular substrates, especially for Gram-negative microorganism with more potent antimicrobial efects, such as Acinetobacter baumannii, Staphylococcus aureus, and P. aeruginosa [66]. Te melatonin mechanisms of the removal of free radicals, the induction of antioxidant enzymes, or the modulation of immunity might protect minks against diseases [67].

Mink Immunity and Resistance to Pseudomonas aeruginosa.
In our previous study [68], we used melatonin and dexamethasone with the dosages of 10 mg and 5 mg, respectively, per body weight (kg) to treat minks at the ages of 5∼6 months before the infections of 1 × 10 9 colony forming units (CFUs) P. aeruginosa. After 20-hour infections, the immunity and resistance results of the minks' infected group (IG) showed more swelling in lungs (Figure 1(a)) with more histopathological changes (Figure 2). Te swelling and histopathological changes reduced with melatonin pretreatment (IGM), but the severity of them increased with less resistance to P. aeruginosa if dexamethasone (IGD) was used for the pretreatment (Figures 1(a) and 2). Similarly, lung lesion and histopathology after 48 h infections (Figure 1(b)) were kept consistent with the results after 24 h infections but were more serious (Figure 1(a)). Long et al. [12] found the dispersion of P. aeruginosa antigen within pulmonary cells and its drift in the lung parenchyma using the immunofuorescence method. After 60-hour infections, survived minks showed the macrophage infltration into limited pulmonary lesions [12].
We also found that bacterial loads of minks after 20-hour infection were higher than those after 8-hour and 48-hour infections (Figures 3(a) and 3(b)). Te bacterial loads of minks in IGM groups were signifcantly lower (P < 0.05) than those in IG groups at the infected time points of 8, 20, and 48 hours (Figure 3(a)); however, the opposite results of bacterial load were found in IGD groups that were significantly higher than IG groups (Figure 3(b)).

Experimental Animal Model for P. aeruginosa Study.
Otani et al. established an experimental model of nonbacteremic pneumonia with a virulent strain of P. aeruginosa in guinea pigs; the lesions were characterized by dissemination of multiple purulogranulomatous changes. In the early and later stages of infections, infltration of polymorphonuclear neutrophils (PMNs) in the bronchiolar and alveolar spaces was difused, where multifocal accumulation with the formation of central spherical grains enclosed bacterial colonies, granulation tissues consisting of large mononuclear cells, fbroblasts, and collagen fbers were developed around the PMN accumulation [69]. P. aeruginosa possessed diferent receptors such as two PA-IL lectins (α-d-galactose (PA-IL) and l-fucose (PA-IIL)) with best characteristics that bound to carbohydrates; such lectin-carbohydrate interactions may create bacterial adherence to epithelial and endothelial cells to cause microbial pathogenicity [70,71]. Kirkeby et al. suggested that minks should be considered as a suitable model to study P. aeruginosa adherence based on the following results: (1) both PA-IL lectins adhered to seromucinous glands were located in the submucosa of the larger bronchi in the lungs;

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PA-IL reacted with the capillaries in the alveolar walls to form the vasa vasorum around the larger vessels with the small blood vessels, but PA-IIL marked the goblet cells in the bronchial surface epithelium; (3) both PA-IL lectins bound to the epithelium in the excretory ducts in the pancreas; (4) PA-IL strongly stained the pancreatic capillaries, but PA-IIL staining was found in the apical part of acinar cells in the exocrine part of the gland, while no lectin reaction was found in the endocrine cells [71]. In addition, Kirkeby et al. suggested that mink is also suitable for the study of P. aeruginosa mediated rhino-sinusitis [13].

Implications and Perspectives
Due to P. aeruginosa infections, high mortality and morbidity and the increased resource utilization and cost appear to infuence the life quality of patients [72][73][74]. Mink is the only known animal that is susceptible to acute, contagious, and fatal lung infections, i.e., hemorrhagic pneumonia as an acute and fatal disease caused by P. aeruginosa, but its pathogenesis has not yet been issued. Salomonsen et al. [4] clarifed the carriers of P. aeruginosa on the nasal mucosa in Danish minks during the season for hemorrhagic pneumonia and illustrated that predisposing factors in the mink itself probably played the key role in disease developments [4]. P. aeruginosa characteristics could afect the innate and adaptive immunizations of lung epithelial cells diferentially in the mediator production and the recruitment of additional immune cell subsets [75]. Curran et al. [75] discussed the quick adaptation of P. aeruginosa to the host microenvironment through the modulation of expressions of cell surface molecules and virulence factors that infuenced the innate and adaptive immune responding efciencies of hosts directly. Such P. aeruginosa interactions with host cells assisted in efective eradication of P. aeruginosa from the most vulnerable mink populations using innovative approaches, such as bacteriophage, immunomodulatory agent, pyocin, QS inhibitor, neutralizing antibody, and aptamer with conventional antibiotic therapy, to reveal the targeted pathways in the combined therapies for clinical cure and survival improvements. To avoid cross-resistance against current therapeutic agents, Wagner et al. suggested the novel mode action antibiotics. Importantly, antivirulence drugs 6 Veterinary Medicine International are expected to yield a signifcantly reduced rate of resistance developments. However, the combined therapy containing antivirulence agents could pave the way toward novel treatment against P. aeruginosa [76].

Conclusions
In summary, this review concludes the useful and valuable information of mink, infection, immunity, and resistance to P. aeruginosa from the previous studies that are crucial for understanding fatal pathogenic infectious pneumonia including the characterization of P. aeruginosa, P. aeruginosa infections, immunity and resistance to P. aeruginosa, and the further implication and perspective in minks, which may contribute to further related research and clinical treatment of P. aeruginosa for mink fatal pathogenic infectious pneumonia and to avoid fatal hemorrhagic pneumonia spreading based on further collaborative international multidisciplinary eforts using current knowledge and strategies.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Jiangsong Bai and Xiao Wang contributed equally to this manuscript.