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

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 [1‐8]. 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 [16‐21]. 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 community-acquired infection [23]. As a pathogen P. mirabilis acquires many virulence determinants that enable it to establish successful infections [24‐26]. A lot of information concerning antibiotic resistance is available for P. mirabilis [27‐35]. 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.

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, extended-spectrum 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 3.2.1.86), Beta N-acetyl-glucosaminidase (nagZ, beta-hexosaminidase) (EC 3.2.1.52), and Glucan endo-1, 4-beta-glucosidase (EC 3.2.1.-) in P. mirabilis SCDR1 suggests that it can hydrolyze chitosan to glucosamine [60‐62]. 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 cm²). 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-resistant Staphylococcus aureus (MRSA), methicillin -resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus (VRE). In addition, these studies showed that Silvercel wound dressing prevented and disrupted the formation of bacterial biofilms [64, 65]. However, this was not the case with our P. mirabilis SCDR1 isolate. Similarly, Sorbsan Silver wound dressing which is made of the fiber of the calcium salt of the alginic acid that contains 1.5% silver [66‐68] did not show any antimicrobial effect against P. mirabilis SCDR1 isolate. Likewise, Colactive® Plus Ag, which is a silver impregnated collagen-based /alginate foam sheet wound dressing, did not show any antimicrobial effect against P. mirabilis SCDR1 isolate. In addition, Exsalt ®SD7 is a silver wound dressing that uses silver oxysalts technology. Silver oxysalts offer greater oxidation states of silver* (Ag²⁺, Ag³⁺) 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 Gram-negative 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-3-phosphate 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 (free-floating 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 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 S-transferases 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 S-transferase 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 two-component 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.

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.