sphingomonas is a group of gram - negative , rod - shaped , non - spore - forming , chemoheterotrophic , strictly aerobic bacterium that produces yellow or off - white pigmented colonies .
the distinctiveness of sphingomonas lies in its possession of ubiquinone 10 as its major respiratory quinone , presence of glycosphingolipids ( gdls ) in their cell envelopes , and its metabolic versatility.1 the genome of sphingomonas is approximately 3,948 kb and contains 70 structural rnas .
it encodes for approximately 3,914 proteins.2 
 sphingomonas utilize glucose as its primary source of carbon .
however , it can also utilize a wide variety of other sugars such as arabinose , fucose , glactose , lactose , mannose , melibiose , sucrose , trehalose , and xylose .
moreover , sphingomonas can also degrade polysaccharides , and many of the strains were also able to utilize one or more of the contaminants as their source of carbon.2 the capability of sphingomonas to utilize a wide range of organic compounds , and to grow and survive under low - nutrient conditions has resulted in its widespread distribution in various environments , including drinking water , soil , air , sinks , shower curtains , and corroding copper pipes .
in addition , sphingomonas paucimobilis has also been found in several clinical specimens , which include hospital water supplies ; temperature probe respirators ; stocked distilled water ; blood ; removes ; hospital dialysis equipment ; patients with meningitis , septicemia , bacteremia , and peritonitis ; and wound infections.3 reported cases of nosocomial infections caused by s. paucimobilis are rarely serious and could be effectively treated with antibiotics . on the contrary , some other reports have concluded that s. paucimobilis nosocomial infections have the ability to severely threaten immune - compromised or ill patients causing health problems with consistent exposure to the source of infection such as shower curtains.4 for example , in 2007 , reports concluded that s. paucimobilis was the cause of bacteremia outbreak in the hemato / oncology units in glhane military hospital in ankara , turkey .
the reports also showed that the clinical isolates were traced back neither to the health care workers nor to the environmental isolates.5 moreover , it was strongly documented that s. paucimobilis created significant problems in various clinical settings , being the most widespread cause of nosocomial infections including bacteremia / septicemia caused by contaminated solutions such as distilled water , hemodialysis fluid , and sterile drug solutions .
cases of pseudo - bacteremia have been recorded in association with s. paucimobilis , as have many cases of unusual infections both invasive and severe , eg , septic arthritis and osteomyelitis.6 moreover , s. paucimobilis caused bloodstream infection in a patient with down syndrome .
it was thereby concluded that s. paucimobilis should be recognized as a nosocomial infectious agent in patients with down syndrome and immunosuppressive disorders.7 in addition , it was also reported that s. paucimobilis has the ability to cause infections in both previously healthy and immune - compromised children8 and can act as a causal agent of osteomyelitis in an immune - competent patient.9 frequent s. paucimobilis infections were observed among our diabetic foot ulcer patients ( 23% of observed gram - negative infections ) .
s. paucimobilis general infection rate was 9.5% , falling just behind staphylococcus aureus ( unpublished data ) .
there is still an ongoing debate about the clinical virulence of s. paucimobilis with a possible conclusion that its clinical importance can not be neglected .
henceforth , this study employs comparative genomics and bioinformatics techniques in order to investigate the pathogenic potentials of sphingomonas spp .
partial 16s rdna sequences of selected pathogenic bacteria , namely , s. paucimobilis ( d16144.1 ) , bacillus anthracis ( gq280034.1 ) , bartonella bacilliformis ( af442955.1 ) , brucella sp .
( ab379686.1 ) , campylobacter jejuni ( ay621112.1 ) , clostridium difficile ( hm245939.1 ) , corynebacterium sp .
( ay034821.1 ) , legionella pneumophila ( nr_041742.1 ) , neisseria meningitidis ( af059671.1 ) , pseudomonas sp .
( jn626189.1 ) , vibrio cholerae ( z21856.1 ) , yersinia pestis ( aj232235.1 ) , were all acquired from the genbank .
these sequences were then aligned using the bioedit built - in clustal w program ( gap opening penalty = 10 , gap extension penalty = 5 , delay divergent sequences = 40% ) .
the resulting alignments were compared , and the final alignments were improved manually and prepared in fasta and mega formats using format converter tool v2.2.5 available online at http://www.hiv.lanl.gov/content/sequence/format_conversion/form.html . in order to establish the phylogenetic relationships among taxa , tree was constructed using the maximum likelihood ( ml ) method based on the tamura 
nei model.10 the percentage of trees in which the associated taxa clustered together is shown next to the branches . initial tree(s ) for the heuristic search was(were ) obtained automatically by applying neighbor - joining and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood ( mcl ) approach , and then the topology was selected with superior log - likelihood value .
the tree was drawn to scale , with branch lengths measured in the number of substitutions per site .
evolutionary analyses were conducted in mega6.11 virulence genes sequences and functions , corresponding to different major bacterial virulence factors of selected pathogens , were collected from genbank and validated in virulence factors of pathogenic bacteria database at http://www.mgc.ac.cn / vfs/. supplementary table 1 shows the tested major pathogenic virulence factors .
selected gene sequences were tested against available sphingomonas gene information using sphingomonas nucleotide blast tool available at http://blast.ncbi.nlm.nih.gov/blast.cgi?page_type=blastsearch&prog_def=blastn&blast_prog_def=megablast&blast_spec=microbialgenomes_13687&db_group=allmg .
the search set all sphingomonas complete genomes , and selected organism was sphingomonas ( tax i d : 13687 ) .
the program selected for the search was blastn , optimizes for fairly similar sequences because of evolutionary divergence of the tested and query taxa .
partial 16s rdna sequences of selected pathogenic bacteria , namely , s. paucimobilis ( d16144.1 ) , bacillus anthracis ( gq280034.1 ) , bartonella bacilliformis ( af442955.1 ) , brucella sp .
( ab379686.1 ) , campylobacter jejuni ( ay621112.1 ) , clostridium difficile ( hm245939.1 ) , corynebacterium sp .
( ay034821.1 ) , legionella pneumophila ( nr_041742.1 ) , neisseria meningitidis ( af059671.1 ) , pseudomonas sp .
( jn626189.1 ) , vibrio cholerae ( z21856.1 ) , yersinia pestis ( aj232235.1 ) , were all acquired from the genbank .
these sequences were then aligned using the bioedit built - in clustal w program ( gap opening penalty = 10 , gap extension penalty = 5 , delay divergent sequences = 40% ) .
the resulting alignments were compared , and the final alignments were improved manually and prepared in fasta and mega formats using format converter tool v2.2.5 available online at http://www.hiv.lanl.gov/content/sequence/format_conversion/form.html . in order to establish the phylogenetic relationships among taxa , tree was constructed using the maximum likelihood ( ml ) method based on the tamura 
nei model.10 the percentage of trees in which the associated taxa clustered together is shown next to the branches . initial tree(s ) for the heuristic search was(were ) obtained automatically by applying neighbor - joining and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood ( mcl ) approach , and then the topology was selected with superior log - likelihood value .
the tree was drawn to scale , with branch lengths measured in the number of substitutions per site .
virulence genes sequences and functions , corresponding to different major bacterial virulence factors of selected pathogens , were collected from genbank and validated in virulence factors of pathogenic bacteria database at http://www.mgc.ac.cn / vfs/. supplementary table 1 shows the tested major pathogenic virulence factors .
selected gene sequences were tested against available sphingomonas gene information using sphingomonas nucleotide blast tool available at http://blast.ncbi.nlm.nih.gov/blast.cgi?page_type=blastsearch&prog_def=blastn&blast_prog_def=megablast&blast_spec=microbialgenomes_13687&db_group=allmg .
the search set all sphingomonas complete genomes , and selected organism was sphingomonas ( tax i d : 13687 ) .
the program selected for the search was blastn , optimizes for fairly similar sequences because of evolutionary divergence of the tested and query taxa .
in this study , the presence of the major known bacterial virulence factors in sphingomonas spp .
was examined . in order to decide on the accurate common pathogenic bacterial species for comparison with sphingomonas spp .
, a phylogenetic tree using the ml method was constructed using partial 16s rdna sequences of selected pathogenic bacteria as mentioned above ( fig .
the phylogenetic tree showed that the selected bacterial species are divided into two major clades ( groups ) , namely , gram - positive bacteria and gram - negative bacteria . the gram - positive bacterial group contained clostridium spp .
, corynebacterium sp . , and bacillus spp . , whereas the gram - negative bacterial group contained the remaining bacterial species
. these results also agree with the known taxonomic arrangement of the tested bacterial species with the exception of bartonella spp .
more importantly , the 16s rdna phylogenetic tree showed brucella sp . to be the closest bacterial taxon to sphingomonas with a bootstrap value of 87 followed by helicobacter spp .
, pseudomonas sp . , and then legionella sp . based on the suggested phylogenetic relationships , the following bacterial species which include brucella sp .
table 1 presents the five selected bacterial genera with its corresponding species , the selected hosts , and the diseases caused by these pathogens .
the selected pathogens were mainly human pathogens having also the ability to infect mammals , protozoa ( legionella sp . ) , and plants ( pseudomonas syringae ) .
all the virulent factors acquired by these pathogens ( table 2 ) were tested for their presence in sphingomonas genomic information .
the major categories of bacterial virulence factors such as adherence , endotoxin , mobility , secretion systems , quorum sensing , and many others are shown in ( table 2 ) .
table 3 and figure 2 present the shared virulence factors among sphingomonas and the selected five bacterial pathogens .
shares the genes responsible for intracellular survival ability ( cgs , manc , and pgm ) with brucella sp . with e - values ranging from 0 to 3.00e  09.1214 in addition , sphingomonas spp .
shares the genes encoding for type iv secretion system such as bmeii0026 with brucella sp . with e - value of 6.00e  04 and identity similarity of 90% . on the contrary , sphingomonas spp . shared no virulence factors with helicobacter spp . or campylobacter sp .
, despite their close phylogenic relationship when compared to pseudomonas sp . and legionella sp .
the sodb encodes for superoxide dismutase , which is a cytoplasmic iron superoxide dismutase important for intracellular survival and transmission.15 regardless of the phylogenetic divergence between sphingomonas spp . and
, it was observed from our results that they shared several major virulence factors such as adherence , antiphagocytosis , iron uptake , proteases , quorum sensing , and others . with regard to adherence , they shared 20 genes , including flgk with e - value of 1.00e  04 and identity similarity of 94% , and flgj with e - value of 6.00e  05 and identity similarity of 89% .
flagella plays an important role as a virulence factor such as in swimming motility toward the infection site and a role in biofilm formation and other pathogenic adaptations.1620 type iv pili play an important role in adherence by assisting the pathogens to attach with their host cells and causing a twitching motility that allows the bacteria to move along the cell surface , and in biofilm formation.17,2125 both sphingomonas spp . and pseudomonas sp .
shared many genes implicated in type iv pili biogenesis and mechanical function of pili , such as pilf with e - value of 4.00e  05 and identity similarity of 97% , and pilb with e - value of 3.00e  04 and identity similarity of 93% .
they shared 10 alginate genes , including algj with e - value of 7.00e  04 and identity similarity of 94% , and algz with e - value of 5.00e  05 and identity similarity of 91% .
the production of alginate permits pathogenic bacteria to form biofilm and contributes to the persistence of bacteria in the lung by acting as an adhesin , which prevents the bacteria from being expelled from the infection site , and the alginate slime layer makes it more difficult for phagocytes to ingest and kill the bacteria.2630 another important bacterial virulence factor shared between sphingomonas spp . and pseudomonas sp . is quorum sensing .
. showed acquiring of both rhll and rhlr with e - values of 3.00e  4 and 5.00e  9 , respectively .
, quorum sensing consists of two separate but interrelated systems , namely , las and rhl , which are found to regulate the production of multiple virulence factors and are also crucial for proper biofilm formation.3133 it is also worth mentioning that both sphingomonas spp . and pseudomonas sp . share seven genes encoding for xcp secretion system ( type ii secretion system ) , including xcpx and xcpr with e - values of 1.00e  5 and 3.00e  174 , respectively .
the xcp secretion system is found to be responsible for secretion of toxins and enzymes into the extracellular fluid.34,35 it was also observed that both sphingomonas spp . and pseudomonas sp . shared many genes involved in iron uptake using both pyochelin ( 10 genes ) and pyoverdine ( 4 genes ) .
the pyochelin is effective at promoting iron uptake in pseudomonas aeruginosa , catalyzes the formation of tissue - damaging free radicals , and also binds other transition metals ( eg , mo(iv ) , co(ii ) ) with appreciable affinity , and is also implicated in the delivery of both co(ii ) and mo(iv ) to p. aeruginosa cells.36,37 the pyoverdine is effective at acquiring iron from transferrin and lactoferrin .
moreover , pyoverdine is cytotoxic because of its ability to stimulate the production of reactive oxygen species.38,39 interestingly , sphingomonas spp . and
pseudomonas sp . shared only one gene responsible for toxin production , namely , plch that is responsible for degrading the phospholipids surfactant , which functions to reduce the surface tension so that the alveoli do not collapse completely when air leaves them during breathing .
it is noteworthy that sphingomyelin ( plch is a multifunctional enzyme ) has been isolated from pseudomonas aeruginosa in 2003.40,41 our comparative analysis was further extended to search for other bacterial toxins that sphingomonas spp .
table 4 shows other toxins that were found to be shared between sphingomonas spp . and
b. pertussis is a strict human pathogen causing whooping cough , a highly contagious respiratory disease marked by severe , spasmodic coughing episodes.42 it was also observed that sphingomonas spp .
contains genes for invasive adenylate cyclase / hemolysin , cyclolysin secretion protein , which is a bifunctional toxin harboring both adenylate cyclase and hemolytic activities , and functions primarily as an anti - inflammatory factor.4345 moreover , sphingomonas spp . contains genes responsible for pertussis toxin and its secretion system , which assists in the attachment of b. pertussis to ciliated respiratory cells , important immunogen and activate cyclic adenosine phosphate ( camp ) , histamine sensitizing factor ( hsf ) , lymphocytosis promoting factor ( lpf ) , islet - activating protein ( iap ) ; interferes with leucocyte function ; and is hemolytic.46,47
contains several major virulence factors , mainly resembling those of pseudomonas sp . other virulence factors from legionella sp .
these findings suggest horizontal gene transfer of virulence factors rather than sharing a common pathogenic ancestor . 

we selected sphingomonas spp . to test its potentiality for being potential virulent pathogen.we tested the phylogenetic relationship of sphingomonas spp . against known virulent pathogens.we screened the presence of various virulent factors from different virulent pathogens in sphingomonas spp.sphingomonas spp .
contains several major virulence factors , mainly resembling those of pseudomonas sp . we selected sphingomonas spp . to test its potentiality for being potential virulent pathogen .
we screened the presence of various virulent factors from different virulent pathogens in sphingomonas spp .
 there is an ongoing debate about the clinical significance of sphingomonas paucimobilis as a virulent bacterial pathogen . in the present study , we investigated the presence of different virulence factors and genes in sphingomonas bacteria . 
 we utilized phylogenetic , comparative genomics and bioinformatics analysis to investigate the potentiality of sphingomonas bacteria as virulent pathogenic bacteria . the 16s ribosomal rna gene ( 16s rdna ) 
 phylogenetic tree showed that the closest bacterial taxon to sphingomonas is brucella with a bootstrap value of 87 followed by helicobacter , campylobacter , pseudomonas , and then legionella . 
 sphingomonas shared no virulence factors with helicobacter or campylobacter , despite their close phylogenic relationship . in spite of the phylogenetic divergence between sphingomonas and pseudomonas , they shared many major virulence factors , such as adherence , antiphagocytosis , iron uptake , proteases , and quorum sensing . in conclusion , sphingomonas spp . 
 contains several major virulence factors resembling pseudomonas sp . 
 , legionella sp . , brucella sp . , and bordetella sp . 
 virulence factors . 
 similarity of virulence factors did not match phylogenetic relationships . 
 these findings suggest horizontal gene transfer of virulence factors rather than sharing a common pathogenic ancestor . 
 sphingomonas spp . is potential virulent bacterial pathogen . 
Introduction
Materials and Methods
Phylogenetic relationships reconstruction
Comparative genomics and bioinformatics analysis
Results and Discussion
Conclusion
Supplementary Material