Detection of ica A and icaD genes and biofilmformation in Staphylococcus spp . isolated from urinary catheters at the University Hospital of Tlemcen ( Algeria )

Staphylococcus spp. is a major cause of infections associated with urinary catheterization and other medical devices. Biofilm formation is an important step in the pathogenesis of these Staphylococci and depends on the expression of the icaADBC operon involved in the synthesis of a polysaccharide intercellular adhesion. In this study, forty-four (44) Staphylococcus spp. obtained from urinary catheters at the University Hospital of Tlemcen (North-West Algeria) were analyzed to investigate the presence or absence of the intercellular adhesion icaA and icaD genes, using the polymerase chain reaction (PCR). Phenotypic biofilm formation was examined by tissue culture plate (TCP) and Congo red agar (CRA) methods. Seventeen (17) of 44 isolates were shown to carry ica-specific DNA, 18 produced slime on CRA plates but only eight produced biofilm spontaneously on the polystyrene surfaces, under normal growth conditions. Upon induction by sugar, biofilm formation could be stimulated in seven (7) of nine (9)ica positive. Biofilm-negative isolates indicated that the icaADBC expression was down-regulated in these strains. Staphylococcus strains isolated from urinary catheters showed high levels of resistance to penicillin (98%) and gentamicine (75%). The data obtained shows the important role of ica-genes, the phenotypic variability of biofilm formation and the multi-resistance to antibiotics as virulence factors of staphylococcus spp. from urinary catheters.It confirms the complexity and diversity of regulation mechanisms implicated in biofilm formation.


Staphylococcus
spp, commensal microorganisms routinely found on the human skin and in the hospital environment (Chokr et al., 2007), are most often associated with chronic infections related to implanted medical devices (Espinasse et al., 2010).
Urinary tract infections can also be caused by these organisms and occur preferentially in patients carrying indwelling urinary tract catheters (Singhai et al., 2012).Urinary catheters have become the second most frequently used medical devices inserted into the human body.Over 40% of nosocomial infections involve the urinary tract, especially in catheterized patients (Holá et al., 2010).
Certain Staphylococcus spp.strains are able to form biofilms on polymer surfaces and it is suggested that this property contributes significantly to the pathogenesis of staphylococcal infection (Cho et al., 2002).Biofilms are a population of multilayered cells growing on a surface and enclosed in the exopolysaccharide matrix.(Cafiso et al., 2004) The development of a biofilm is considered to be a twostep process.First, the bacteria adhere to a surface mediated by a capsular antigen, namely the capsular polysaccharide/adhesin (PS/A), then the bacteria multiply to form a multilayered biofilm, with production of polysaccharide intercellular adhesin (PIA) which mediates cell to cell adhesion (Nasr et al., 2013;EL Farran et al., 2013) and provides the protection against opsonophagocytosis and antimicrobial peptideactivity (Spiliopoulou et al., 2012) The synthesis of PIA is encoded by the products of the chromosomal ica-genes (intercellular adhesion), which are organized in an operon structure.The operon contains the ica ADBC genes, in addition to the ica Rgene which exerts a regulatory function and is transcribed in the opposite direction.Once this operon is activated, four proteins are transcribed, IcaA, IcaD, IcaB and IcaC, which are necessary for the synthesis of PIA (Cafiso et al., 2004;Atshan et al., 2012;Agarwal and jain, 2013;Mertens and Ghebremedhin, 2013).PIA is synthesized from UDP-N-acetylglucosamine by N-acetylglucosaminyltransferase which is encoded by the ica locus, particularly icaA.
The expression of this gene alone induces low enzymatic activity and production of low amount of polysaccharide.However, the simultaneous expressions of icaA and icaD promote a significant increase in Nacetylglucosaminyltransferase, with a con-sequent increase in the amount of polysaccharide, hence forming oligomers of 10-20 b-1,6-Nacetylglucosamine residues (Dobinski et al., 2002;Gotz, 2002;Oliveira et al., 2010 ;Gad et al., 2012;Namvar et al., 2013).IcaB is the deacetylase responsible for the deacetylation of mature PIA.In addition, the transmembrane protein IcaC seems to be involved in the externalization and elon-gation of the growing polysaccharide (Diemond-Hernández et al., 2010).
The expression of the ica operon, and as a result the formation of biofilms, seems to be highly variable among staphylococci.Thus, biofilm formation is influenced by the environmental signals and can be induced in response to external stress and subinhibitory concentrations of certain antibiotics (Ziebuhr et al., 1997;Mempel et al., 1994;Cho et al., 2002;Mertens and Ghebremedhin, 2013).
The differentiation of staphylococci with respect to their biofilm phenotype might help to elucidate the impact of staphylococci for the diagnosis of infections associated with biomedical devices.These observations can be useful in the prevention of device-related infections (Mathur et al., 2006).
Several studies have been published on the detection of the Ica gene among the staphylococci strains (Cho et al., 2002;Chaieb et al., 2005;Touati et al., 2007;Gad et al., 2009;Wang et al., 2010;Duran et al., 2010;Nasr et al., 2013).However, despite the increasing interest in the subject in recent years, data collection from medical institutions in Algeria are relatively difficult, hence the low number of related studies.
The objective of the present study was to characterize Staphylococcus spp.strains isolated from urinary catheter at the University Hospital of Tlemcen, in terms of their antibiotic susceptibility, biofilm formation and presence of icaA and icaD genes.

Bacterial strains
The strains used in this study were isolated from urethral catheterization from the Intensive Care Unit, Urology and Neurology Services at the University Hospital Center (CHU) of Tlemcen (North-West Algeria).Urinary catheters were removed aseptically from patients suffering from a catheter-related urinary tract infection.The urinary catheters under study are Latex probes not impregnated with antibiotics, transported at 4°C, and immediately analyzed at the laboratory.

Identification
After removal of the catheter, the microbiological analysis was performed using the Brun-Buisson technique (Brun-Buisson, 1994), which consists in rinsing the catheter lumen with saline water and vortexing its intravesical end before cultivation on Chapmanagar medium which allows the selection of staphylococci.
Moreover, all isolates were identified by classic microbiological methods including colony morphology, Gram staining, catalase test, coagulase test and the Api-Staph test (BioMérieux®).

Tissue culture plate method (TCP)
Quantitative determination of biofilm formation on 96-well tissue culture plates (Sigma, UK) was performed based on the Christensen method (Christensen et al., 1985), with a modification in the incubation length which was extended to 48 h.Therefore, the biofilm production was evaluated in three different media, namely Brain Heart Infusion Broth (BHIB), BHIB with 2% sucrose, and BHIB with 1% glucose.
The bacteria were grown overnight in respective media, and the cultures were then diluted 1:100 and incubated in a microtiter polystyrene plate at 37°C.Microtiter wells were washed three times with distilled water, dried in an inverted position, and stained with 0.5% (w: v) crystal violet solution (Mathur et al., 2006).The adherent cells were resuspended in 95% ethanol solution and the absorbance was measured at 540 nm with a micro ELISA auto reader (model 680, Biorad, UK).The isolates were classified into three categories: a) non adherent, optical density lower than 0.120; b) weakly adherent, optical density greater than 0.120 and smaller or equal to 0.240, c) strongly adherent, optical density greater than 0.240.

Congo red agar method (CRA)
The Congo red test was performed as previously described by Freeman et al. (1989).The medium consisted of brain heart infusion broth (BHIB, 37 g/L), sucrose (50 g/L), agar no.1 (10 g/L) and Congo red stain (0.8 g/L).Congo red was prepared as a concentrated aqueous solution and autoclaved at 121°C for 15 min, separately from the other medium constituents and was then addedto the mixture when the agar had cooled to 55°C.The plates were inoculated and incubated aerobically for 24 to 48 h at 37°C.
Biofilm producers form black colonies on CRA, whereas nonproducers form red colonies.The Congo red dye directly interacts with certain polysaccharides, forming colored complexes (Jain and Agarwal, 2009).

Detection of icaA and icaD loci
Extraction of bacterial DNA was performed by thermal shock.After overnight culture on Luria Bertani agar plates (Bio-Rad, Marnes-la-Coquette, France), 5 colonies were suspended in 500 ml of DNase and RNase-free water (Invitrogen, England).The suspension was boiled at 100°C for 10 min in a thermal block (Polystat 5, French), then centrifuged at 15000 rpm for 5 min.An aliquot of 2 µL of the supernatant was used as DNA template for PCR.

Characterization of staphylococcal isolates froma urinary catheter
A total of 44 strains were collected from a urinary catheter used more than 48 h at the University Hospital of Tlemcen.After biochemical analysis, all 44 strains were identified as staphylococcal species and included: 21 S. epidermidis, 11 S. saprophyticus, 11 S. aureus, and 1 S. hominis.

Study of biofilm production by tissue culture plate (TCP) method
Quantitative determinations of biofilm formation were carried out by measuring the adherence of broth cultures to 96-well tissue culture plates, as outlined in materials and methods.Under standard growth conditions in BHIB, only 8 (18%) out of 44 isolates were capable of forming a biofilm.
To evaluate the impact of environmental growth conditions on biofilm formation by clinical isolates, we performed some biofilm assays using growth media supplemented with 1% glucose or 2% sucrose, as previouslydescribed.This resulted in an increase in the number of isolates capable of biofilm formation; 15 (34%) isolates out of 44 were able to produce a biofilm, in the presence of one of these media supplements.Thus, the overall rate of biofilm-forming strains rose from 18 to 34% after stimulation (Figure 1).

Detection of icaA andica D loci by PCR
The PCR technique was applied to all 44 staphylococcal strains.The icaA and icaD genes were detected concomitantly in 17 (38.5%) of the 44 staphylococcal isolates.Furthermore, 2 of them presented the loci icaDonly.

Relationships between the presence of the ica operon, slime production and the TCP method
Sixteen (16) of the 17 icaA and icaD positive strains were found to be slime producers and 8 produced a visible biofilm on polystyrene surfaces under standard growth conditions.After stimulation by sugar supplementations, 7 out of 9 of the previous icaA/icaD positive and biofilmnegative strains formed a visible biofilm on polystyrene tissue culture plates.
All 23 icaA/icaDnegative strains were unable to produce slime on CRA and biofilm on polystyrene tissue culture plates.The results obtained with all the strains are summarized in Table 1.

Antibiotic sensitivity testing
Antibiotic susceptibility testing showed that most of Staphylococcus spp.strains were resistant to more than nine antibiotics and were found to be susceptible to four major antibiotics: Rifampim, Fosfomycin, Clindamycin and Chloramphenicol.Moreover, no strains were found to be vancomycin-and pristinamyci-resistant.Biofilm-producing strains were found to be more resistant compared to non-producing ones.
The total percentage of resistance against each antibiotic and the relationship between biofilm formation and antimicrobial resistance pattern are represented in Table 2.

DISCUSSION
In the last two decades, with the increasing use of indwelling medical devices, nosocomial infections caused by Gram-positive bacteria, in particular staphylococcus spp., have become more prevalent as a cause of hospital-acquired infection (Fitzpatrick et al., 2002).
The major pathogenic factor is the ability to produce an extracellular slime and form a biofilm, thus making the clinical treatment extremely difficult.The biofilm development process requires polysaccharidic intercellular adhesin, which is synthesized by the enzymes encoded by the intercellular adhesion cluster (ica) (Martín-López et al., 2002).
Early detection and management of biofilm-forming staphylococci can be one of the essential steps towards the prevention and management of device-associated nosocomial infections (Nasr et al., 2013).
In our study, 44 staphylococcus spp.were isolated from a urinary catheter in order to test the occurrence of slime genes, biofilm production and slime production in staphylococci using PCR, TCP and Congo red agar methods, respectively.
The results reveal that S. epidermidis are the most frequently isolated species, corresponding to 48% of all strains.Other staphylococcus species were also identified, including S. saprophyticus, S. aureus and S. hominis.These results are close to those obtained by Diemond-Hernández et al. (2010).
It has been noticed in several studies that the S. epidermidis is the most frequently isolated speciesin nosocomial infections and is the most common causative organism found in infections of implanted medical devices.It makes up a significant part of the normal bacterial flora of the human skin and mucous membranes and is probably easily introduced as a contaminant during the surgical implantation of the polymeric device (Otto, 2008).
In this study, icaA and icaD were detected concomitantly in 17 of the 44 staphylococcus spp.strains isolated from a urinary catheter and an icaA-/icaD+ profile was found in two strains.
These results are close to those obtained by Cafiso et al. (2004) who investigated the presence of genes involved in biofilm production and found that 35% of isolates were positive for the icaA and icaD genes and some of them carried the icaD gene only.
In the TCP assay, with BHIB used as standard growth medium, 8 of 17 icaA/D positive strains exhibited a biofilm.This is in agreement with the observations of other investigators (Cho et al., 2002;Mathur et al., 2006;Johannes et al., 2002) who found that few or no biofilmproducing isolates could be detected using this medium.Surprisingly, supplementation of BHIB medium with different sugars (BHIB 2%suc , BHIB 1%glu ) increased biofilm formation, and 34% of the studied isolates formed a biofilm in at least one of the used media.Furthermore, two isolates of staphylococci ica D+/ica A-did not form a biofilm in both media.
These observations suggested that biofilm formation in staphylococcus spp is strongly dependent on growth conditions, and indicated that the use of various sugar supplementations is essential for biofilm formation (Mathur et al., 2006).
Moreover, the expression of the ica operon and therefore the formation of biofilms seems to be highly variable among staphylococci (Ziebuhr et al., 1997;Mempel et al., 1994).Thus, the biofilm expression is influenced by environmental signals and can be induced in response to external stress and subinhibitory concentrations of certain antibiotics (Cho et al., 2002).Cramton et al. (2001) suggested that anaerobiosis strongly increases biofilm expression.The expression of a biofilm is also regulated by iron, with a maximum expression occurring at low concentrations (Chaieb et al., 2005).
However, two staphylococcal strains icaA+ and icaD+ remained biofilm negative even under PIA-expressionstimulating growth conditions.The detection of no biofilm, despite the presence of ica, could be due to several reasons such as the inactivation of the ica operon by insertion of an IS256 element in the icaC gene (Ziebuhr et al., 1999), the action of the icaR repressor (Conlon et al., 2002), or the post-transcriptional regulation (Dobinsky et al., 2003).
Comparison of the CRA test and the results obtained by PCR revealed that among the 17 icaA+ and icaD+ strains, 16 were slime-producers.In fact, these results  (2006) reported this phenomenon and suggested that in these strains; variability in theica locus sequence exists, allowing the production of a polysaccharide which reacts with the anti-PIA serum.
Furthermore, Staphylococcus spp.isolated from urinary catheters showed high levels of resistance to different classes of antibiotics except vancomycine and pristinamycin.They were significantly resistant to penicillin (98%), oxacilline (79%), gentamicine (75%) and ofloxacine (73%).These results are close to those obtained by Touati et al. (2007) who reported that Staphylococci isolated from catheter-related infections are significantly resistant to oxacilline (76.8%), gentamicine (46.4%) and ofloxacine (75%).Biofilm-forming strains which express ica genes are more resistant to antibiotics.This result confirms that a biofilm adds to the virulence profile of Staphylococcus strains isolated from urinary catheters.Biofilms constitute a reservoir of pathogens which are associated with the resistance to antimicrobial agents and cause chronic infections (Seif El-Din et al., 2011;Khan et al., 2011).
The multicellular organization of a bacterium in biofilms gives them the advantage to acquire new genes.The biofilm is a perfect medium for the exchange o fresistance plasmids (Touati et al., 2007) as it combines both the greater probability of contact between cells and the negligible effect of shear forces (Donlan, 2001).Gilbert et al. (2002) reported that biofilm producers are 10-1000 times less susceptible to antibiotics than are the equivalent cells growing planktonically.A biofilm hampers the penetration of an antimicrobial and the concentrations required to eradicate biofilm-producing bacteria are higher than those required to eradicate strains that did not produce biofilm (Seif El-Din et al., 2011).
In conclusion, our findings show the significant role of ica genes as a virulence marker for Staphylococcal isolates.Their association with biofilm-forming strains strongly suggests that expressions of icaA and icaD genes play a role in the pathogenetic mechanisms o finfections associated with urinary catheters.Hence, in infections caused by biofilm-producing staphylococci, the differentiation with respect to biofilm phenotype might help to modify the antibiotic therapy and preventinfections related biomedical devices.

Table 1 .
relationships between the presence of the ica operon and biofilm production.

Table 2 .
The relation between biofilm formation and antimicrobial resistance pattern.