Detection of biofilm formation , icaADBC gene and investigation of toxin genes in Staphylococus spp . strain from dental unit waterlines , University Hospital Center ( UHC ) Tlemcen Algeria

The formation of biofilms on pieces of equipment and dental chair unit waterlines has attracted more interest and concern. This study aimed to assess the reliability of two phenotypic methods used to detect the presence of biofilms and the icaADBC operon, and identify various toxin genes by multiplex polymerase chain reaction (PCR) in clinical staphylococcal isolates. A total of thirty-one (31) staphylococcal strains were isolated from waterline tubings in the dental unit. Two phenotypic methods were used for the detection of biofilm production. PCR detected the presence of the ica operon, and a multiplex PCR assay for the detection of staphylococcal toxin genes was done. Biofilm production was detected in twenty-six (83.87%) isolates by tissue culture plate method (TCP). The CRA method indicated that twenty (64.51%) of these strains could produce slime. By using the ica operon test, the genotypic ability to form biofilm in was identified in fourteen (45.16%) strains. Five (16.12%) strains of Staphylococcus warneri harbored one or more toxin genes detected by PCR multiplex. The presence of ica operon gene does not always have relationship with in vitro biofilm formation and genes encoding the staphylococcal toxin. Among the strains studied, few of them had the ica operon, toxin gene, and phenotypic ability to form a biofilm at the same time.


INTRODUCTION
Since their introduction, water cooled rotating instruments in dental units have been highly contaminated by microorganisms.These are not only micro-organisms from the main water supply, but also those which are sucked back from the patient's mouth.They adhere to the internal surfaces of water-carrying pipes and form strong adherent biofilms (Lee et al., 2001).The development of biofilms is facilitated by the ready adhesion of bacteria to the hydrophobic polymeric plastic tubing material used in dental equipment and by the low flow rate and the intermittent patterns of use of water with overnight and weekend stagnation (Decoret et al., 2005).However, some pathogenic or opportunistic bacteria are also found: Pseudomonas aeruginosa, Serratia marcescens, Legionella pneumophila, Mycobacterium spp and Staphylococcus spp.(Barbeau et al., 1996;Michel and Just, 1984;Pankhurst and Coulter, 2007;Singh and Coogan, 2005;Szymanska et al., 2008).Over the last *Corresponding author.E-mail: meriemlachachi@yahoo.fr.few years, several studies have been done to elucidate the structures and pathogenetic mechanisms by which staphylococci are able to cause severe and irreducible infections associated with biomaterials (An and Friedmann , 1998;Foster and Mcdevitt , 1994).
The principal component of a biofilm is a polysaccharide intercellular adhesin {PIA} (Lappin-Scott and Bass, 2001;Ziebuhr et al., 1997).PIA is composed of a beta-1,6-N-acetylglucosamine polymer synthesized by an enzyme codified by the ica operon found on the bacterial chromosome, that includes a regulating element of four genes (A, B, C, and D), and a transposable element, IS256 (Kozitskaya et al., 2004).It is known that the A gene codifies the N-acetylglucosamyl transferase enzyme responsible for synthesizing PIA.This enzyme is not very active in vitro, but co-expression of the D gene increases the activity.IcaB is the deacetylase responsible for the deacetylation of mature PIA and the transmembrane protein IcaC seems to be involved in the externalization and elongation of the growing polysaccharide (Lappin-Scott and Bass, 2001;Gerke et al., 1998).The organism has an array of cell surfaces and secreted virulence factors that allow it to cause illnesses (Lowy, 1998).They include the classical staphylococcal enterotoxins (sea, seb, sec and sed), SEs and SEls (seh, selk, sell, selm, selo, selp, selq and ser) and the toxic shock syndrome toxin-1 (tst) (Jarraud et al., 2002;Holtfreter et al., 2007).Most of the genes encoding these toxins are located on mobile genetic elements such as bacteriophages, pathogenicity islands (SaPIs), genomic islands,and plasmids (Holtfreter and Broker, 2005;Baba et al., 2002).The purpose of the present study was to determine the biofilm producing ability and the presence of the icaADBC gene and characterize the toxin genes in Staphylococci isolated from waterline tubings in the dental unit of the University Hospital Center (CHU) of Tlemcen in Algeria, as well as to assess the reliability of two phenotypic methods used for the detection of biofilms; namely the Congo red agar and the Microtiter plate methods.

Bacterial strains
The strains of Staphylococcus spp.were isolated from 13 waterline tubings from different dental chairs (air/water syringes, ultrasonic scalers, conventional handpieces) used in the dental unit of the University Hospital (UHC) of Tlemcen in Algeria, and analyzed in the laboratory immediately.The isolates were initially identified by standard microbiological techniques including Gram stain,catalase and coagulase tests as well as the Api-Staph test (Biomerieux).

Tissue culture plate method (TCP)
Quantitative measurement of biofilm production in Staphylococcus spp.strains was detected using a microtiter assay.In brief, cells were grown overnight in BHI with 2% saccharose (BHISuc).Next, the solution was diluted 1:100 and inoculated into microtiter plates.After 24 h of incubation at 37°C the plates were washed, stained with crystal violet and the optical density was measured at 570 nm (Christensen et al., 1985).The experiment was performed in triplicate and repeated three times.For individual strains of Staphylococcus spp were classified into three categories: weakly adherent, optical density < 0.120, Moderately adherent, optical density between (0.120 and 0.240), strongly adherent, optical density > 0.240 (Mathur et al., 2006).

Congo red agar method (CRA)
The Congo red agar method was used for detecting the ability to produce slime, and the CRA medium was prepared with 37 g/L BHI broth, 50 g/L sucrose, 10 g/L agar, and 0.8 g/L Congo red.The Congo red stain was prepared as a concentrated aqueous solution and autoclaved at 121°C for 15min separately from other medium constituents, and was then added when the agar had cooled to 55°C.Plates were inoculated and incubated at 37°C for 24 h.A positive result was indicated by black colonies whereas non producing strains developed red colonies (Freeman et al., 1989).

Detection of the ica operon
Genomic DNA from Staphylococcus spp.strains was extracted by the technique of thermal shock from a fresh culture after 18 to 24 h at 37°C on plain agar.Several colonies were suspended in 500 µl of ultra-pure water (molecular biology, DNase and RNase-free) and brought to the Boiling Point (100°C) for 10 min and then transferred directly into ice (0°C) for 5 min.An amount of 300 µl of the supernatant is then recovered after centrifugation at 14500 tpm for 10 min.Next, the supernatant containing the DNA was stored at -20°C.
The primer sequence, amplification conditions and product length for icaA, icaB, icaC and icaD of Staphylococcus spp.are presented in Table 1.PCR was performed in a DNA thermal cycler (Applied biosystems PCR system2700).The reaction was done in a 25 µl volume containing the above-mentioned primers (1 mM each), together with 150 ng of the extracted DNA, 100 mM of each of dATP, dCTP, dGTP, and dTTP, 1 U of Taq DNA polymerase, and 10 mM PCR buffer (pH 9.0).The magnesium concentration in the mixture was 3 mM for each gene.After amplification, 10 µl of the PCR mixture was analyzed by agarose gel electrophoresis (2% agarose in Tris-borate-EDTA) (Paluch-Oles et al., 2011).
The primers used in this study, have been previously described by (Jarraud et al., 2002;Holtfreter et al., 2007) and are also used by the National Reference Center (CNR) of staphylococci in the city of Lyon in France.

RESULTS
A total of 31 strains of coagulase-negative staphylococci  In the TCP method, sixteen (51.61%) isolates were strong producers, ten (32.25%) were moderate and five (16.12%) were weak biofilm producers (Figure 1) Phenotypic production of slime by all the strains under study was assessed by culture on CRA plates.A number of twenty isolates (64.51%) were slime-producing, and eleven (35.48%) were non-slime-producing.
By using the ica operon test, the genotypic ability to form biofilm was identified in fourteen (45.16%) strains of the thirty-one staphylococcal isolates, among fourteen strains possess the ica operon ,ten strains were positive for icaD, two strains were positive for icaB, one strain  were positive for icaBD, and only one isolates carried the icaADB genes , all strains studied didn't harbred the icaC gene.
The results of biofilm production by staphylococci using the TCP (quantitative), CRA (qualitative) methods, and the presence of the icaADBC genes are shown in Table 2. Thirteen of the fourteen isolates carried the ica operon were positive by the TCP method and ten (32.25%) by the CRA method.

Multiplex polymerase chain reaction (PCR) for detection of selected staphylococcal genes
To substantiate the multiplex PCR technique, thirty-one strains of Staphylococcus spp. that were tested by multiplex PCR were also screened for the presence of individual toxin genes, using the method described above.Out of these 31 strains, only five strains of Staphylococcus warneri carried toxin genes, three (9.67%) were positive for edinABC, three (9.67%) for seh, three (9.67%) for sem, and one (3.22%)contained the gene for Luk M.Among these five isolates harbored the toxin genes, three were positive for ica operon , and four were able to produce a biofilm by TCP and slime by CRA.(Table 3).

DISCUSSION
It is well known that dental unit waterlines are heavily populated with bacteria.The lumen of the small bore tubes are colonized by a tenacious freshwater biofilm that acts as a reservoir for opportunistic pathogens (Barbeau ).In the present study, a total of 31 staphylococcal strains were isolated from the dental unit waterline tubings.The TCP assay is most widely used and is considered as a standard test for detection of biofilm formation (Mathur et al., 2006;Gad et al., 2009).This method has been reported to be the most sensitive, accurate and reproducible screening method for the determination of biofilm production by clinical isolates of staphylococci and has the advantage of being a quantitative tool for comparing the adherence of different strains (Christensen et al., 1985;Mathur et al., 2006).
Together with its capacity to examine a large number of isolates simultaneously, was the reason for its use in our study.TCP method could detect 83.86% of staphylococcal isolates to be biofilm producers which could be further classified as 51.61% strong producers and 32.25% moderate producers.This method gave the best discrimination between strong, moderate producers and non-producers of biofilms (Bose et al., 2009).In the study of Mathur et al. (2006), 57.8% of staphylococcal clinical isolates displayed a biofilm-positive phenotype and 14.47 and 39.4% exhibited high and moderate biofilm formation, respectively; while 46% were weak isolates with no biofilm detected.In another study, of the 50 staphylococcal isolates, biofilm production was found in 46%, with different intensities determined by TCP method and 6% were strong producers, 12% were moderate and 8% were weak biofilm producers; while in 54% no biofilm was formed (Nasr et al., 2012).
For staphylococcal species, the two possible explanations of the ability to colonize artificial materials are the bacterial production of polysaccharide slime and the presence of adhesins for the host matrix proteins that, in vivo, are adsorbed onto the biomaterial surface (Barth et al., 1989;Cristhensen et al., 1982;Montanaro et al., 1998).
When comparing the results of both CRA and TCP methods, 64.51% were biofilm producers by CRA while 83.86% were positive by TCP .The CRA method was found to be easier and faster to perform than other phenotypic methods but could probably identify only the strong biofilm producers.It is imprecise in the identification of moderately biofilm producing strains.Hence, it could detect the least number of biofilm producers (Samant Sharvari and Pai Chitra, 2012).
Of the fourteen staphylococcal strains found positive for the ica operon, 10 were biofilm producers by the CRA method.In the present study, it is also shown that, four very red strains were found positive for ica operon.These data suggest that the phenotypic change may be influenced by the environmental conditions (Chaieba et al., 2005).
The expression of the icaADBC operon has recently been shown to be a highly variable factor modulated by phase variation and genome rearrangements.It was suggested that the variable biofilm expression contributes to adaptation of the bacterium in changing the environmental conditions of incubation (Costerton et al., 1995;Rachid et al., 2000;Ziebuhr et al., 2000).
Some authors attributed the absence of biofilm production in some staphylococcal isolates, despite the presence of the ica operon, to the insertion of a 1332-bp sequence element, known as IS256, in icaA causing its inactivation (Ziebuhr et al., 1997;Cho et al., 2002;Kiem et al., 2004) .However, the transposition of IS256 into the ica operon has been found to be a reversible process, as after repeated passages of the PIA negative insertional mutants, the biofilm-forming phenotype could be restored (Ziebuhr et al., 1999).
These same authors concluded that CRA might be imprecise in the identification of positive isolates when compared to molecular analysis of the genes involved in biofilm production.Among the thirty-one strains of Staphylococcus spp tested by multiplex PCR, a total of five (16.12%) isolates were positive for one or more toxin genes.Of these five strains, two (6.45%) possessed the gene for icaB and one (3.22%)was positive for icaD.In another study of one hundred and seven (107) isolates tested, twenty-one (19.6%) were positive for sea, twentysix (24.3%) were positive for the TSST-1 gene (tst), six (5.6%) for seb, eight (7.5%) for sec, and two (1.9%) contained the gene for sed.It is important to recognize that this technique will identify only strains harboring the toxin genes and is independent of the expression and secretion of the toxin (Mehrotra et al., 2000).
The results showed that four strains produce the biofilm by TCP and slime by CRA carried the toxin genes.Other examples exist in which secreted toxins may play a role in biofilm formation, (Caiazza and O'Toole., 2003 ) demonstrated a role for alpha-hemolysin in Staphylococcus aureus biofilm formation, and in particular, this toxin appears to be required for cell-to-cell interactions, and they were initially surprised to find that a secreted toxin had such a dramatic impact on biofilm formation.Table 3 shows the relation between the phenotypic methods for the detection of biofilm production, the presence of the ica opreron and toxin genes.

Conclusion
The present results demonstrated the presence of the ica operon, toxin genes and biofilm production, though about half of staphylococci isolated from waterline tubings in the dental unit were capable of forming biofilms, the presence of the icaADBC gene was not always associated with in vitro formation of a biofilm.Comparing the phenotypic biofilm detection methods, CRA was easier and faster to perform.Data from this study indicate that TCP is an accurate and reproducible method for screening and it can be a reliable quantitative tool to determine biofilm formation by clinical isolates of staphylococci.Most notably the system of this multiplex PCR reliably, rapidly, and simultaneously detects each of the toxin genes.These strains which secrete toxins and have the genes responsible for biofilm formation are responsible for many nosocomial infections.It's a common belief that long-term exposure to the bacterium and the toxins they carry could sensitize the lungs and present a risk to patients and dental personnel.

Figure 1 .
Figure 1.Quantification of biofilms formed by strains of Staphylococcal in microplate for 24 h in BHI saccharose + 2% by measuring OD570 nm.Biofilm formation was quantified by crystal violet.Values are means ± SD of three independent experiments.

Table 1 .
Primers sequence and amplification conditions for PCR of ica genes.

Table 2 .
Association between results of CRA, TCP and presence of ica operon in staphylococcal strains.

Table 3 .
Association between results of toxin genes and presence of ica operon and production biofilm in Staphylococcus spp strains isolated from waterline tubing in the dental unit.