Genome sequencing and analysis of the first spontaneous Nanosilver resistant bacterium Proteus mirabilis strain SCDR1

Background P. mirabilis is a common uropathogenic bacterium that can cause major complications in patients with long-standing indwelling catheters or patients with urinary tract anomalies. In addition, P. mirabilis is a common cause of chronic osteomyelitis in Diabetic foot ulcer (DFU) patients. We isolated P. mirabilis SCDR1 from a Diabetic ulcer patient. We examined P. mirabilis SCDR1 levels of resistance against Nanosilver colloids, the commercial Nanosilver and silver containing bandages and commonly used antibiotics. We utilized next generation sequencing techniques (NGS), bioinformatics, phylogenetic analysis and pathogenomics in the characterization of the infectious pathogen. Results P. mirabilis SCDR1 was the first Nanosilver resistant isolate collected from a diabetic patient polyclonal infection. P. mirabilis SCDR1 showed high levels of resistance against Nanosilver colloids, Nanosilver chitosan composite and the commercially available Nanosilver and silver bandages. The P. mirabilis -SCDR1 genome size is 3,815,621 bp. with G + C content of 38.44%. P. mirabilis-SCDR1 genome contains a total of 3533 genes, 3414 coding DNA sequence genes, 11, 10, 18 rRNAs (5S, 16S, and 23S), and 76 tRNAs. Our isolate contains all the required pathogenicity and virulence factors to establish a successful infection. P. mirabilis SCDR1 isolate is a potential virulent pathogen that despite its original isolation site, the wound, can establish kidney infection and its associated complications. P. mirabilis SCDR1 contains several mechanisms for antibiotics and metals resistance, including, biofilm formation, swarming mobility, efflux systems, and enzymatic detoxification. Conclusion P. mirabilis SCDR1 is the first reported spontaneous Nanosilver resistant bacterial strain. P. mirabilis SCDR1 possesses several mechanisms that may lead to the observed Nanosilver resistance. Electronic supplementary material The online version of this article (10.1186/s13756-017-0277-x) contains supplementary material, which is available to authorized users.


Background
The production and utilization of nanosilver is one of the primary and still growing applications in the field of nanotechnology. Nanosilver is used as the essential antimicrobial ingredient in both clinical and environmental technologies. Nanosilver is utilized in the formulation of dental resin amalgams, medical device coatings, water filter antimicrobial coating, antimicrobial agents in air sanitizers, textiles, pillows, respirators, socks, wet wipes, detergents, soaps, shampoos, toothpastes, washing machines, bone cement, wound dressings, hospital beds and furniture to control infection and support anti-biofilm activity [1][2][3][4][5][6][7][8]. Nanosilver is known to exert inhibitory and bactericidal effects against many Gram-positive, Gram-negative and fungal pathogens [9]. Latest studies suggest that the use of nanosilver-containing wound dressings prevents or reduces microbial growth in wounds, and may improve the healing process [10]. Moreover, antibacterial nanosilver-containing wound dressing gels may be important for patients that are at risk of non-healing of diabetic foot wounds and traumatic/surgical wounds [11].
Increased usage of nanosilver in both medical and environmental products has generated concerns about the development of bacterial resistance against the antimicrobial ingredient. Bacterial resistance against metallic silver has been documented with several bacterial strains such as E. coli Enterobacter cloacae, Klebsiella pneumoniae and Salmonella typhimurium [12,13]. However, information about bacterial resistance against Nanosilver is scarce. Only Gunawan et al., (2013) reported the occurrence of induced adaptation, of non-targeted environmental Bacillus species, to antimicrobial Nanosilver [14]. In this study, we report on a spontaneous nanosilver-resistant Proteus mirabilis isolate ("SCDR1"). Proteus mirabilis is a motile gram-negative bacterium that is characterized by its swarming behavior [15,16]. P. mirabilis is a common uropathogen that can cause major complications. In addition, P. mirabilis can cause respiratory and wound infections, bacteremia, and other infections [16][17][18][19][20][21]. In fact, P. mirabilis is a common cause of chronic osteomyelitis in Diabetic foot ulcer (DFU) patients along with Bacteroides fragilis, E coli, and Klebsiella pneumoniae [22]. Generally, P. mirabilis is responsible for 90% of genus Proteus infections, and can be considered as a communityacquired infection [23]. As a pathogen P. mirabilis acquires many virulence determinants that enable it to establish successful infections [24][25][26]. A lot of information concerning antibiotic resistance is available for P. mirabilis [27][28][29][30][31][32][33][34][35]. P. mirabilis is intrinsically resistant to tetracyclines and polymyxins. Moreover, multidrug-resistant (MDR) P. mirabilis strain resistance to β-lactams, aminoglycosides, fluoroquinolones, phenicols, streptothricin, tetracycline, and trimethoprim-sulfamethoxazole has been reported [36]. However, limited information about heavy metals, including silver, is available. In this study, we present the first report and genome sequence of the nanosilver resistant bacterium P. mirabilis strain SCDR1, isolated from diabetic foot ulcer (DFU) patient.

Bacterial isolate
Proteus mirabilis strain SCDR1 was isolated from a diabetic ulcer patient in the diabetic foot unit at the University Diabetes Center, King Saud University. P. mirabilis SCDR1 was the first nanosilver resistant isolate to be collected from a diabetic patient's polyclonal infection. A Proper wound swab was obtained from the patient and was sent for further microbiological study and culture. Wounds needing debridement were debrided before swabbing the surface of the wound. The specimen was inoculated onto blood agar (BA; Oxoid, Basingstoke, UK) and MacConkey agar (Oxoid) and incubated at 37°C for 24-48 h. The Vitek 2 system and its advanced expert system were used for microbial identification, antibiotic sensitivity testing, and the interpretation of results. ID GN cards were used to identify the bacterial isolate, and AST-N204 was used for the antimicrobial susceptibility testing of gram-negative rods. Manual disk diffusion and MIC method for AgNPs and antibiotic sensitivity testing were performed when required. Results were categorized according to EUCAST 2.0 VITEK 2 MIC breakpoints.

Disk diffusion antimicrobial susceptibility testing
Disk diffusion antimicrobial susceptibility testing was performed as described by Matuschek et al. [38]. Briefly, Mueller-Hinton (MH) agar plates were inoculated with agar with an inoculum corresponding to a McFarland 0.5 turbidity with a sterile cotton swab to prepare bacterial lawns of the abovementioned bacterial test strains. Sterile discs were loaded with different concentrations (50-200 ppm) of colloidal silver nanoparticles solutions and the Nanosilver chitosan composite (composite concentration ranged from 0.1% and 0.01 M to 3.2% and 0.16 M from chitosan and Silver nitrate respectively) and then placed on Mueller-Hinton (MH) agar plates with bacterial lawns. Within 15 min of application of antimicrobial disks, the plates were inverted and incubated at 37 ∘ C for 16 h. All experiments were done in aseptic conditions in a laminar air flow cabinet. After incubation, inhibition zones were read at the point where no apparent growth was detected. The inhibition zone diameters were measured to the nearest millimeter. Similarly, 5 mm desks from the commercially available bandages were prepared in aseptic conditions and tested for antimicrobial activity, as previously described.

Minimum bactericidal (MBC) and minimal inhibitory concertation (MIC) test
MBC and MIC testing were performed as described by Holla et al., [39]. Briefly, a dilution with 1 × 10 5 CFU/ml (equivalent to 0.5 McFarland) was used as an inoculum for MIC testing. Different volumes that contained a range of silver Nanoparticles (50-700 ppm) were delivered to 7.5 ml of Muller-Hinton (MH) broth, each inoculated with 0.2 ml of the bacterial suspensions. Within 15 min of application of silver nanoparticles, the tubes were incubated at 37 ∘ C for 16 h in a shaker incubator at 200 rpm. We included a positive control (tubes containing inoculum and nutrient media without silver nanoparticles) and a negative control (tubes containing silver nanoparticles and nutrient media without inoculum).

Biofilm formation
In order to test the ability of P. mirabilis SCDR1 isolate to form biofilm, a culture was prepared by inoculation on Columbia agar, supplemented with 5% blood and incubated at 37 C°for 24 h. The culture was then used to prepare 0.5 McFarland standard bacterial suspension. Wells of sterile 96-well flat-bottomed plastic microplates were filled with 250 μL of the Brain-heart infusion broth. Negative control wells contained the broth only. Twenty μL of bacterial suspension were then added to each well. The plate was incubated at 37 C°for 24 h. Following the incubation, the content of each well was aspirated and washed three times with 300 μL of sterile distilled water. The remaining attached bacteria were fixed with 200 μL of methanol per well, and after 15 min the plates were emptied and left to dry air. After this, the plates were stained for 5 min with 160 μL per well of crystal violet used for gram stain. Excess stain was rinsed off by placing the microplates under running tap water. After the plates were air dried, the dye which was bound to the adherent cells was re-solubilized with 160 μL of 33% (v/v) glacial acetic acid per well. The optical density (OD) was measured at 570 nm [40].
Molecular genomics analysis DNA purification, sequencing, bioinformatics and phylogenetic analysis DNA isolation, purification, genome sequencing, bioinformatics and phylogenetic analysis were performed as described by Saeb et al., 2017 [41]. In addition, we used Mauve [42] and CoCoNUT [43] to generate the whole genome pairwise and multiple alignments of the draft P. mirabilis strain SCDR1 genome against selected reference genomes. Furthermore, we performed whole genome phylogeny based proteomic comparison among P. mirabilis SCDR1 isolate and other related Proteus mirabilis strains using Proteome Comparison service which is a protein sequence-based comparison using bi-directional BLASTP available at (https:// www.patricbrc.org/app/SeqComparison) [44].
The heavy metal resistance gene search for P. mirabilis SCDR1 contigs were investigated using PGAAP and PATRIC gene annotation services, PATRIC Feature Finder searches tool and BacMet (antibacterial biocide and metal resistance genes database) available at (http:// bacmet.biomedicine.gu.se/) [44,51].
P. mirabilis SCDR1 isolate showed high resistance against colloidal and composite Nanosilver and metallic silver compared with other tested Gram positive and negative bacterial species. For instance, Table 1, shows the resistance of P. mirabilis SCDR1 against colloidal Nanosilver assessed by the disk diffusion method, in comparison with S. aureus ATCC 29213, P. aeruginosa ATCC 27853, E. coli ATCC 25922 and E. cloacae ATCC 29212. Generally, P. mirabilis SCDR1 showed high resistance (0.0 cm), while K. pneumoniae showed the highest sensitivity (1.5-1.9 cm) against all tested silver nanoparticle concentrations (50-200 ppm). S. aureus also showed high sensitivity (1.4-1.6 cm) against all tested silver nanoparticle concentrations. None of the tested bacterial isolates except for P. mirabilis SCDR1 showed any resistance against silver-nanoparticles, even against the lowest concentration (50 ppm). Furthermore, Table 2 shows the resistance of P. mirabilis SCDR1 against colloidal Nanosilver assessed by a minimal inhibitory concentration method, compared with other tested Gram positive and negative bacterial species. Once more, P. mirabilis SCDR1 showed high resistance against the gradually increased concentrations of colloidal nanosilver. We observed P. mirabilis SCDR1 bacterial growth to colloidal Nanosilver concentration up to 500 ppm. On the other hand, K. pneumoniae showed the highest sensitivity against silver nanoparticles, with no observed growth at only 100 ppm colloidal nanosilver concentration. In addition, both E. coli and P. aeruginosa showed high sensitivity against silver nanoparticles, with no observed growth at 150 ppm colloidal Nanosilver concentration. Conversely, S. aureus tolerated only 200 ppm colloidal Nanosilver concentration. Similarly, Table 3 shows the resistance of P. mirabilis SCDR1 against silver and Nanosilver composite assessed by disk diffusion method. Nanosilver chitosan composites, with a concentration ranging from between 0.1% and 0.01 M to 3.2% and 0.16 M from chitosan and Silver nitrate respectively, had a comparable killing effect on both Gram positive and negative bacterial, namely S. aureus and P. aeruginosa. Meanwhile, none of the tested Nanosilver chitosan composites had any killing effect on P. mirabilis SCDR1. Similarly, all the tested commercially available silver and Nanosilver containing wound dressing bandages showed the enhanced killing effect on both S. aureus and P. aeruginosa. However, all these wound dressing bandages failed to inhibit P. mirabilis SCDR1 growth. P. mirabilis SCDR1 was able to produce strong biofilm with OD of 0.296.

General genome features
Data from our draft genome of P. mirabilis SCDR1 was deposited in the NCBI-GenBank and was assigned accession number LUFT00000000. The bacterial bioinformatics database and analysis resource (PATRIC) gene annotation analysis showed the presence 308 unique genes of the biosynthesis of secondary metabolites such as tetracycline, Streptomycin, Novobiocin, and Betalain. It is also noteworthy that Xenobiotics Biodegradation and Metabolism pathways also maintained a high number of dedicated unique gene (245) (Additional files 1 and 2: Tables S1 and S2).

Pathogen identification and phylogenetic analysis
As previously stated, biochemical identification of the isolate confirmed the identity of our isolate as belonging to the Proteus mirabilis species. Moreover, Primary analysis of Metaphlan showed that Proteus mirabilis is the most dominant species in the sample (Fig. 1). The appearance of other bacterial species in the Metaphlan diagram is explained by the genomic homology similarity of other bacteria to Proteus mirabilis. P. mirabilis SCDR1 genome showed high similarly, 92.07%, to the genome of P. mirabilis strain BB2000 followed by P. mirabilis strain C05028 (90.99%) and P. mirabilis strain PR03 (89.73%) ( Table 4). A similar scenario was observed when constructing the phylogenetic relationship between our isolate and other Proteus mirabilis available in the NCBI-GenBank. 16Sr DNA-based maximum likelihood phylogenetic tree (Fig. 2) showed that our isolate is located within a large clade that contains the majority of Proteus mirabilis strains and isolates. In addition, P. mirabilis SCDR1 was shown to be closely related to the reference strain P. mirabilis HI4320 compared with P. mirabilis BB2000, which is located in another clade of four Proteus mirabilis taxa. On the contrary, the whole genome Neighbor-joining phylogenetic tree of Proteus mirabilis spices including P. mirabilis SCDR1 isolate (Fig. 3), showed that our isolate was more closely related to P. mirabilis BB2000 compared with the reference strain/genome P. mirabilis HI4320. However, Fig. 4 showed that P. mirabilis SCDR1 exhibited obvious genetic divergence from other Proteus mirabilis species. Similar results were observed when performing pairwise pair-wise whole genome alignment of P. mirabilis strain SCDR1 against reference genomes (Fig. 4). This was also confirmed with the clear divergence among P. mirabilis SCDR1 Proteus mirabilis species at the proteomic level (Fig. 5).

Bacterial pathogenic and virulence factors
Pathogenomics analysis using PathogenFinder 1.1 showed that our input organism was predicted as a human pathogen, and the probability of being a human pathogen was 0.857. P. mirabilis SCDR1 comparative proteome analysis showed 35 matched hits from pathogenic families and only one hit from non-pathogenic families (Additional file 3: Table S3). In addition, genome analysis showed that P. mirabilis SCDR1 isolate contains numerous virulence factor genes and/or operons that marks it out to be a virulent pathogenic bacterium.
These virulence factors include swarming behavior, mobility (flagellae), adherence, toxin and hemolysin production, Urease, Quorum sensing, iron acquisition systems, proteins that function in immune evasion, cell invasion and biofilm formation, stress tolerance factors, and chemotaxis related factors (Additional file 4: Table S4).

Proteus Mirabilis SCDR1 Resistome Antibiotic resistance
Antibiotic resistance identification Perfect and Strict analysis using Resistance Gene Identifier (RGI) showed that P. mirabilis SCDR1 isolate contains 34 antibiotic resistance genes that serve in 21 antibiotic resistance functional categories (Additional file 5: Table S5 and Fig. 6). Meanwhile, using the less strict (Loose) antibiotic resistance identification criteria identified 3750 hits in P. mirabilis SCDR1 genome that represent potential AROs (Antibiotic Resistance Ontology) that fall into 59 antibiotic resistance functional categories (Fig. 7) of which 38 are considered to lose antibiotic resistance functional categories. Modified loose antibiotic resistance identification criteria, by removing all hits with objectionable e-values, lead to a number of 366 antibiotic resistance related hits (Additional file 6: Table S6 and Fig. 7). Manual genome annotation based mining resulted in the identification of 64 drug resistance related proteins in P. mirabilis SCDR1 genome (Additional file 7: Table S7).

Proteus Mirabilis comparative genomics based resistome analysis
We performed a species-wide antibiotic resistome constituent analysis of P. mirabilis. All available P. mirabilis genomes, including the P. mirabilis SCDR1 genome, were included in this analysis (Table 5). Results of our analysis (Table 6 and Fig. 8) showed that the number of the observed antimicrobial resistance based ontologies (AMRO) in P. mirabilis genomes is 61. Only 16 AMROs were observed amongst all the studied 56 P. mirabilis genomes. Meanwhile, 13 AMROs were observed amongst 55 P. mirabilis genomes. In addition, only four AMROs were observed amongst 54 P. mirabilis genomes and two AMROs were observed amongst 48 P. mirabilis genomes. This suggests that the core constituent of antibiotic resistome of P. mirabilis species is made up of 35 AMROs (Table 6). On the other hand, eight AMROs were detected only in one P. mirabilis genome. For example, the membrane fusion component of tripartite multidrug resistance system was only observed in our P. mirabilis SCDR1 genome.

Discussion
Proteus mirabilis isolate was observed as mixed culture along with S. aureus isolate while testing our produced silver Nanoparticles against several pathogenic S. aureus isolates [9]. Whereas other tested Gram positive and negative bacteria showed great sensitivity against silver Nanoparticles, P. mirabilis, SCDR1 isolate exhibited extreme resistance. P. mirabilis SCDR1 isolate resistant against at least one antibiotic belonging to ansamycins, glycopeptides, fucidanes, cyclic peptides, nitroimidazoles, macrolides, lincosamides, folate pathway inhibitors and aminocoumarin antimicrobial categories. Moreover, our isolate exhibited intrinsic resistance against tetracyclines and polymyxins specific to P. mirabilis species [36,52,53]. However, fortunately, our isolate was sensitive to several operational antimicrobial categories such as penicillins with b-lactamase inhibitors, extendedspectrum cephalosporins, carbapenems, aminoglycosides, fluoroquinolones and phosphonic acids. In addition, our P. mirabilis SCDR1 isolate showed high resistance against colloidal and composite Nanosilver and metallic silver when compared to other tested Gram positive and negative bacterial species, both qualitatively and quantitatively. To the best of our knowledge, this is the first reported case of bacterial spontaneous resistance to colloidal and composite nanosilver. However, Gunawan et al., (2013) reported the occurrence of induced adaptation, of non-targeted environmental Bacillus species to antimicrobial Nanosilver [14]. In addition, it was found that bacteria can straightforwardly develop resistance to AgNPs, and this occurs by relatively simple genomic changes [54]. They both showed that a Bacillus sp. environmental isolate and an E.coli isolate were able to adapt to Nanosilver cytotoxicity upon continued exposure. Nonetheless, as previously stated, P. mirabilis SCDR1 exhibited instantaneous resistance against nanosilver without the need for any prolonged exposure. P. mirabilis SCDR1 demonstrated resistance against colloidal nanosilver assessed either by disk diffusion or by minimal inhibitory concentration methods. While all used concentrations of colloidal Nanosilver have shown strong effects on all tested microorganisms (Table 1), there was no effect on the bacterial growth of P. mirabilis SCDR1 even at the highest used concentration (200 ppm). Similarly, P. mirabilis SCDR1 was able to resist ten fold (500 ppm) higher than K. pneumoniae (50 ppm), five fold higher than P. aeruginosa and E. coli (100 ppm) and two and a half fold (200 ppm) higher than S. aureus and E. cloacae (Table 2). Moreover, while both laboratory prepared and commercially available silver and Nanosilver composite showed a clear effect against both S. aureus and P. aeruginosa, the most common pathogens of diabetic foot ulcer, not effect was observed against P. mirabilis SCDR1 (Table 3). Although chitosan nanosilver composites have documented combined effect against both Gram positive and negative pathogens [37] no effect was observed against P. mirabilis SCDR1. Silver is a highly toxic element for microbes. The Nanosilver exhibits high surface to volume ratio, which shows increased antimicrobial power in comparison to the same bulk silver material [55]. It is suggested that the antimicrobial mechanism of silver ions involves the disruption of phospholipids of cytoplasmic, and the disruption of DNA replication, impairing the function of ribosomes to transcribe messenger RNA and/or inactivation of cytochrome b by binding with sulfhydryl group [56]. P. mirabilis SCDR1 genome analysis showed that our isolate contains a large number of genes (245) responsible for xenobiotics biodegradation and metabolism (Additional file 2: Table S2). Although P. mirabilis SCDR1 does not contain the chitinase genes responsible for Chitin and chitosan degradation, it contained Chitin binding protein (cbp, 203 amino acid protein). This may justify the ability of P. mirabilis SCDR1 to resist the antimicrobial effect of chitosan. Chitin-binding protein even without any catalytic domain can facilitate the degradation of β-chitin by means of disrupting the crystalline chitin polymer structure [57,58]. Microbial ability to produce proteins with high specific affinity to a certain crystalline chitin structure could be pivotal for the capability of bacteria to differentiate and react to specific crystalline chitin structures [59]. In addition, these chitin-binding domains may affect chitin degradation by facilitating adhesion of cells to the chitinous materials [57]. Thus, although we did not detect chitinase genes in P. mirabilis SCDR1, the presence of Chitin-binding protein suggests that P. mirabilis SCDR1 has some mechanisms of protection against chitin and the chitosan antimicrobial effect. In addition, the presence of genes encoding for the members Chitosanase family GH3 of N, N′-diacetylchitobiose-specific 6-phospho-beta-glucosidase (EC 3.  [60][61][62]. This justifies the lack of antimicrobial effect of chitosan against P. mirabilis SCDR1. Likewise, P. mirabilis SCDR1 showed resistance against all the tested commercially available silver and Nanosilver containing wound dressing bandages. These silver containing commercially available bandages (wound dressing material) use different manufacturing technology and constituents. For example, Silvercel wound dressing contains high G calcium alginate in addition to 28% Silver-coated fibers (dressing contains 111 mg silver/100 cm 2 ). The silver-coated fibers encompass elemental silver, which is converted to silver oxide upon contact with oxygen. Silver oxide dissolves in fluid and releases ionic silver (Ag + ) that has antimicrobial action [63]. Clinical studies showed that Silvercel wound dressing is effective against many common wound pathogens, including methicillin- (Ag 2+ , Ag 3+ ) capable of interacting with microbial DNA, proteins and lipids, as well as providing potent oxidizing action through the increased power of Ag2 +,3+ for advanced biocidal activity. Exsalt ®SD7 showed high antimicrobial activity against tested Gramnegative and positive bacteria and fungi tested [69]. P. mirabilis SCDR1 isolate showed high resistance against Exsalt ®SD7. In addition, P. mirabilis SCDR1 isolate showed high resistance against Puracol ® Plus Ag, which is made of 100% Collagens in addition to antimicrobial silver. Furthermore, Actisorb® Silver 220, which is a sterile primary dressing encompassing an activated charcoal cloth, impregnated with silver within a spun bonded perforated nylon sleeve [70] was not active against P. mirabilis SCDR1 isolate. Pathogenomics analysis showed that P. mirabilis SCDR1 isolate is a potential virulent pathogen (Additional files 3 and 4: Tables 3 and 4). P. mirabilis SCDR1 shows that it possesses the characteristic bull's eye pattern of swarming behavior. Presenting swarmer cells form is associated with the increase in expression of virulence genes [71]. Swarming is important to P. mirabilis uropathogenesis. It has been shown that swarming bacteria that move in multicellular groups exhibit adaptive resistance to multiple antibiotics [72]. Swarming behavior promotes the survival of bacteria in harsh environments or in unfavorable conditions. Moreover, migrating swarm cells display an increased resistance to many of antimicrobial agents. Therefore antimicrobial resistance could be a general feature of bacterial multicellular social behavior [73]. For example, the swarm cells of P. aeruginosa were able to migrate very close to the disc containing arsenite, indicating resistance to this heavy metal [73]. It has been suggested that high densities promote bacterial survival, the ability to move, as well as the speed of movement, confers an added advantage, making swarming an effective strategy for prevailing against antimicrobials including heavy metals [72,73]. Furthermore, altruism or self-sacrifice is a suggested phenomenon associated with swarming, which involves risk of wiping out some individuals upon movement of bacteria to a different location, allowing the remaining individuals to continue their quest [72,74]. Another suggested phenomenon associated with swarming is selfish behavior, in which the survival may be highest on top cells that are furthest from the antimicrobial agent while the lower cells in the swarm die because of the proximity to antimicrobial agents [72,75]. Thus, selfish cells within the swarm sense where the best location is to avoid the toxic effect of the antimicrobial agent. Swarming behavior may indeed be one main reason for the observed nanosilver resistance of P. mirabilis SCDR1. Thus, maintaining high cell density, through the observed quorum sensing ability (Additional file 4: Table S4) and the circulation within the multilayered colony to minimize exposure to the heavy metal in addition to the death of individuals that are directly exposed, could be the key to the observed nanosilver resistance.
P. mirabilis SCDR1 isolate exhibited the ability of biofilm formation and also our pathogenomics analysis showed that it contains the genes responsible for this,  such as glpC gene coding for anaerobic glycerol-3phosphate dehydrogenase subunit C (EC 1.1.5.3), pmrI gene coding for UDP-glucuronic acid decarboxylase and baaS gene coding for biofilm formation regulatory protein BssS. We believe that the ability of P. mirabilis SCDR1 to form biofilm may also assist in the observed Nanosilver resistance. Biofilm formation can reduce the metal toxic effect by trapping it outside the cells. It was found that in the relative bacteria Proteus vulgaris XC 2, the biofilm cells showed considerably greater resistance to Chloromycetin compared to planktonic cells (freefloating counterparts) [76]. Moreover, it is suggested that the ability of biofilm formation may play a pivotal role in Polymyxin B antibiotic resistance in P. mirabilis [77]. Furthermore, it was found that biofilm formation is very important for heavy metal resistance in Pseudomonas sp. and that a biofilm lacking mutant was less tolerant to heavy metals [78]. Furthermore, it was found that both Extracellular Polysaccharides and Biofilm Formation is a resistance mechanism against toxic metals in Sinorhizobium meliloti, the nitrogen-fixing bacterium [79]. In addition, several reports claimed that the minimum inhibitory concentration (MIC) of some antibiotics for biofilms can be 1000-fold higher than that for planktonic bacteria [80].
It is well known that there are several mechanisms for metal resistance. These include physicochemical interactions, efflux, intracellular sequestration and extracellular precipitation by the excreted polymeric compounds [79]. Indeed, additional to swarming activity, Polysaccharides and biofilm formation (Additional file 4: Table S4), P. mirabilis SCDR1 contains several genes and proteins that also facilitate metal resistance including silver and Nanosilver (Table 8). Our results indicate the presence (See figure on previous page.) Fig. 4 Pair-wise Whole Genome Alignment of P. mirabilis strain SCDR1 against reference genomes. a P. mirabilis BB200 and P. mirabilis SCDR1 Mauve whole genome alignment, b P. mirabilis HI4320 and P. mirabilis SCDR1, c P. mirabilis AOUC001 and P. mirabilis SCDR1, d P. mirabilis CYPM1 and P. mirabilis SCDR1, e P. vulgaris CYPV1 and P. mirabilis SCDR1, f P. mirabilis SAS71 and P. mirabilis SCDR1 Mauve whole genome alignment of endogenous silver and copper resistance mechanism in P. mirabilis SCDR1. We observed the presence of gene determinants of Copper/silver efflux system, oprB encoding for Copper/silver efflux system outer membrane protein CusC (outer membrane efflux protein OprB), oprM encoding for Copper/silver efflux system outer membrane protein CusC (outer membrane efflux protein OprM), cusC_1 encoding for Copper/silver efflux system outer membrane protein CusC (RND efflux system outer membrane lipoprotein), cpxA encoding for Copper sensory histidine kinase and outer membrane component of tripartite multidrug resistance system (CusC). In addition, we observed the presence of several Copper resistance genes/proteins were detected, namely, copA, copB, copC, copD, cueO, cueR, cutC, cutF and CuRO_2_CopA_like1. A similar endogenous silver and copper resistance mechanism has been described in E. coli and has been associated with the loss of porins from the outer membrane and up-regulation of the native Cus efflux mechanism, which is capable of transporting silver out of the cell [81,82]. However, the genetic basis resistant phenotypes are still not fully known, and it is not known if they are obligatory or sufficient to exhibit resistance to silver [83]. Thus, we suggest a comprehensive study for this endogenous silver resistance mechanism within the Proteus mirabilis as well as E. coli. Furthermore, we observed the presence of genes encoding to enzymes involved in heavy metal resistance such as Glutathione S-transferase (EC 2.5.1.18) (gst1, gst, Delta and Uncharacterized) in P. mirabilis SCDR1 genome. Thus, we propose a role of Glutathione Stransferases of P. mirabilis SCDR1 in the observed Nanosilver resistance. Glutathione S-transferases (GSTs) are a family of multifunctional proteins that play an important role in the detoxification of harmful physiological and xenobiotic compounds in organisms [84]. Moreover, it was found that a Glutathione Stransferase is involved in copper, cadmium, Lead and mercury resistance [85]. Furthermore, it was found that GST genes are differentially expressed in defense against oxidative stress caused by Cd and Nanosilver exposure [85].
Moreover, we observed the presence of a complete tellurite resistance operon (terB, terA, terC, terD, terE, terZ) which was suggested as contributing to virulence or fitness and protection from other forms of oxidative   stress or agents causing membrane damage, such as silver and Nanosilver, in P. mirabilis [86]. Several other heavy metal resistance genes and proteins were observed in the P. mirabilis SCDR1 genome. These included arsM encoding for arsenite S-adenosylmethyltransferase (Methyltransferase type 11), which play an important role in prokaryotic resistance and detoxification mechanism to arsenite [87,88] and merB encoding for alkylmercury lyase that cleaves the carbon-mercury bond of organomercurials, such as phenylmercuric acetate [89]. Moreover, numerous heavy metal resistance proteins were observed, such as magnesium/cobalt efflux protein CorC, metal resistance proteins, nickel-cobalt-cadmium resistance protein NccB, arsenical pump membrane protein (ArsB permease), Lead, cadmium, zinc and mercury transporting ATPase (Table 8).
In order to gain information about antimicrobial resistome constituents in P. mirabilis species, we performed comparative genomics analysis amongst all available 56 P. mirabilis genomes, including the P. mirabilis SCDR1 genome. As stated before, all P. mirabilis genomes shared 16 AMROs (Table 6). For example, all genomes contained the AMRO of copper sensory histidine kinase CpxA in cpxA mutant confer resistant to amikacin, copper-sensing twocomponent system response regulator CpxR, which is a regulator that promotes acrD expression when phosphorylated by a cascade involving CpxA, a sensor kinase and linked to cefepime and chloramphenicol resistance in Klebsiella pneumoniae [90]. However, different P. mirabilis genomes varied in the remaining 45 studied AMRO (Table 6). For example, genomics analysis of P. mirabilis-SCDR1 showed that our isolates contained genetic determinants for fluoroquinolones resistance (gyrA, parC and parE) [91,92], Daptomycin and Rifamycin resistance (rpoB) [93], Chloramphenicol (cpxR, cpxA and cat) [90,94], Ethidium bromide-methyl viologen resistance protein (emrE) [95] and Polymyxin and colistin resistance (phoP) [96]. In addition, several multidrug resistance efflux systems and complexes were observed. These include MdtABC-TolC, which is a multidrug efflux system in Gram-negative bacteria, including E. coli and Salmonella that confer resistance against β-lactams, novobiocin and deoxycholate. It is noteworthy that MdtABC-TolC and AcrD plays a role in metal resistance (copper and zinc), along with their BaeSR regulatory system [97] which was also was found in our P. mirabilis SCDR1 genome [ Table 7], and thus may also play an additional role in silver resistance. MdtABC-TolC contains MdtA, which is a membrane fusion protein, TolC, which is the outer membrane channel and  MdtBC that forms a drug transporter. In the absence of MdtB, the MdtAC-TolC has narrower drug specificity, leading to the loss of novobiocin resistance [98]. The MdtABC and AcrD systems may be related to bacterial metal homeostasis by transporting metals directly. This is to some extent similar to the copper and silver resistance mechanism by cation efflux of the CusABC system belonging to the RND protein superfamily [97,99]. As stated before, this is the first report for spontaneous resistance against nanosilver. However, Gunawan et al., (2013) reported the natural ability of Bacillus sp. to adapt to nanosilver cytotoxicity under prolonged cellular oxidative stress stimulation through the nanoparticles incubation [14]. They found that the induced effects of adaptation continued even after discontinuation of nanosilver exposure. They also suggested that this characteristic ability of the ubiquitously-occurring Bacillus sp. may pose adversative consequences to the extensive use of nanosilver. Moreover, Graves et al., (2015) showed that after 225 generations of the treatment with nanosilver, the treated E. coli populations demonstrated greater fitness compared with control strains in the presence of silver nanoparticles [54]. We could also have suggested that the observed P. mirabilis SCDR1 nanosilver resistance might be an adaptive effect of the use of silver containing bandages in the Diabetic foot ulcer clinic. However, the reference strain P. mirabilis ATCC 29906 (isolated from urogenital tract of Homo sapiens) also showed resistance against colloidal nanosilver. In addition, it seems that nanosilver resistance is a widespread character in P. mirabilis species, since all other tested 50 isolates showed resistance against colloidal nanosilver [unpublished data]. Comparative metal resistance in P. mirabilis genomes contains some of the genetics determinants that can aid in nanosilver/silver resistance [unpublished data]. However, this conclusion needs to be further tested in a larger number of P. mirabilis isolated from different infection sites and geographical locations.
Increasing antimicrobial nanosilver usage could prompt a silver resistance problem in Gram-negative pathogens, particularly since silver resistance is already known to exist in several such species [81,     100]. Both exogenous (horizontally acquired Sil system) endogenous (mutational Cus system) resistance to silver has been reported in Gram-negative bacteria [13,81]. Li et al. [81] selected five Escherichia coli mutants that present a ≥ 64-fold decreases in silver susceptibility compared with their original strain. All the mutants exhibited loss of expression of outer membrane porins (OmpF or OmpF/C), which seemingly resulted in the reduction of outer membrane permeability. These findings implied that reduced silver susceptibility is a result of restricting silver entrance into the bacterial cell. Moreover, they found that these mutants express active efflux that pumps silver outside of the cell. It was found that the cus CFBA operon is the responsible of silver efflux pump. Similarly, in our case, we observed the presence of resistance operon with high similarity to the cus operon, which is a chromosomally encoded system because of the lack of any plasmid in P. mirabilis SCDR1. However, both endogenous and exogenous silver resistance systems, in Gram-negative bacteria, remain incompletely understood [83]. The occurrence of induced nanosilver resistance (in vitro) in Bacillus sp. and E. coli [14,54], spontaneous resistance (in our case) and the frequent uses and misuses of nanosilver-containing medical products should suggest adopting an enhanced surveillance systems for nanosilver-resistant isolates in medical setups. In addition, there should be greater control over utilizing nanosilver-containing products in order to maintain nanosilver as a valuable alternative approach in the fight against multidrug resistant pathogens.

Conclusion
In the present study, we introduced the P. mirabilis SCDR1 isolate that was collected from a diabetic ulcer patient. P. mirabilis SCDR1 showed high levels of resistance against nanosilver colloids, nanosilver chitosan composite and the commercially available nanosilver and silver bandages. Our isolate contains all the required pathogenicity and virulence factors to establish a successful infection. P. mirabilis SCDR1 contains several physical and biochemical mechanisms for antibiotics and silver/nanosilver resistance, which are biofilm formation, swarming mobility, efflux systems, and enzymatic detoxification.