Genes encoding adhesion factors and biofilm formation in methicillin-resistant Staphylococcus aureus in Morocco

Introduction: Infections involving methicillin-resistant Staphylococcus aureus (MRSA) remain a serious threat to hospitalized patients worldwide. MRSA is characterized by recalcitrance to antimicrobial therapy, which is a function not only of widespread antimicrobial resistance, but also the capacity to form biofilms. The present study evaluated the presence of genes encoding adhesion factors and the biofilmforming capacity in MRSA. Methodology: In this study, 53 isolates of MRSA, recovered from December 2010 to May 2014 in a mother and child hospital, CHU Mohamed VI in Marrakech, Morocco, were screened for the presence of bap and ica genes associated with biofilm formation, and for bbp, cna, ebpS, eno, fib, fnbA, fnbB, clfA, and clfB genes that encode microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). The biofilm formation assay was performed in 96-well microtiter polystyrene plates. The presence of genes was determined by polymerase chain reaction (PCR). Results: An association was found between icaD gene detection and biofilm formation; 100% of the strains harbored icaD and produced biofilm. None of the isolates harbored bap or bbp. Furthermore, 96.23% isolates were positive for fnbA, 60.37% for eno, 43.39% for clfA and clfB, 11.32% for cna, 9.34% for ebpS, 5.66% for fib, and 1.89% for fnbA. Conclusions: Our findings showed that the MRSA carriage in Marrakech children was high. The genetic variations of adhesion genes require further investigation.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is among the most prominent of all bacterial pathogens.It is a commensal inhabitant of a significant proportion of the healthy population, but it also has the capacity to cause a diverse array of infections, ranging from relatively superficial skin infections to serious, life-threatening infections, including endocarditis, pneumonia, and osteomyelitis.Many forms of staphylococcal infection are associated with the formation of a bacterial biofilm [1,2].Biofilm is a microbial-derived sessile community characterized by cells that are irreversibly attached to a substratum of interface or to each other, embedded in a matrix of extracellular polymeric substances that they have produced, and exhibiting an altered phenotype with respect to growth rate and gene transcription [3].Two steps appear to be involved in the molecular mechanisms implicated in host colonization by Staphylococcus aureus.The first step is bacterial adhesion to host extracellular matrix and plasma proteins mediated by different proteins of the family of microbial surface components recognizing adhesive matrix molecules.The second step is growth-dependent accumulation of bacteria in multilayered cell clusters where genes involved in biofilm formation come into play [4,5].The microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are protein components of the microbial surface that are able to interact with and bind to a variety of extracellular proteins.Among adhesins, the fibronectinbinding protein, the collagen-binding protein, the elastin-binding protein, and the bone sialoproteinbinding protein have been proven to significantly contribute to tissue colonization in various pathological conditions.Among the ica genes, icaD have a significant role in slime production in Staphylococcus aureus and Staphylococcus epidermidis [6].Biofilm formation protects microorganisms from opsonophagocytosis and antimicrobial agents [7].
In human Staphylococcus aureus strains, several of these virulence factors play a role in life-threatening infections [8]; for this reason, considerable effort has been expended to define the specific staphylococcal factors that promote biofilm formation and/or persistence within a biofilm.The goal of the present study was to evaluate the presence of genes encoding adhesion factors and biofilm forming capacity in MRSA.We further evaluated the correlation between biofilm production by clinical isolates and the presence of icaD genes.

Clinical strains
In this study, 53 isolates of MRSA, 41 strains of blood culture origin and 12 staphylococcal strains of all origins, were recovered from December 2010 to May 2014 in a mother and child hospital, CHU Mohamed VI in Marrakech, Morocco.

Identification
All isolates were identified by classic microbiological methods including colony morphology, mannitol fermentation, Gram staining, catalase test, coagulase test, and the API Staph test (bioMérieux, Marcy l'Etoile -France).Methicillin resistance was confirmed using a cefoxitin disk (30 μg) on Mueller-Hinton agar plates (Bio-Rad, Marnes-la Coquette, France) as recommended by the French Society for Microbiology (FSM, 2013), and polymerase chain reaction (PCR) was used to confirm the presence of the mecA gene.

Biofilm formation assay
The biofilm formation assay was performed in 96well microtiter polystyrene plates as described by O'Toole et al [9].At first, cells were grown on liquid Luria Bertani (Bio-Rad, Marnes-la-Coquette, France) in 96-well microplates.After incubation at 37°C, detached cells were gently rinsed three times with sterile water, and the bacteria that attached to the surface were stained with crystal violet 0.1% for 20 minutes.The excess crystal violet dye was washed by rinsing the wells with water.The amount of biofilm formed was estimated by solubilization of the dye with 95% ethanol, and the optical density (OD) was determined at 540 nm.The absorbance was recorded by microplate reader.The biofilm formation were divided into three categories in this study: the strains with OD 550 < 0.38, 0.38 > OD 550 < 0.70, and OD 550 > 0.70 were defined as biofilm formers of weak level, moderate level, and strong level, respectively, based on the ODs [10].Pseudomonas aeruginosa ATCC 27853 was used as a positive control.

Genomic DNA extraction
DNA templates for the PCR were generated by suspending five colonies in 500 μL of DNase and RNase-free water (Bio-Rad, Marnes-la-Coquette, France).The suspension was boiled at 100°C for 10 minutes in a thermal block (Polystat 5, Illkirch-Graffenstaden, France) and immediately frozen at 0°C for 5 minutes, then centrifuged at 15,000 rpm for 5 minutes.After centrifugation, 300 μL of supernatant was recovered.Supernatant containing DNA was stored at -20°C until further use as a DNA template for PCR [11].

PCR assay on MSCRAMM and biofilm genes
The presence MSCRAMM and biofilm genes were detected by PCR using forward and reverse primers to detect the presence of icaD (intercellular adhesion gene D), bap (encoding biofilm-associated protein), bbp (encoding bone sialoprotein-binding protein), clfA and clfB (encoding clumping factors A and B), cna (encoding collagen-binding protein), ebpS (encoding elastin-binding protein), eno (encoding laminin-binding protein), fnbA (encoding fibronectin-binding protein A), fnbB (encoding fibronectin-binding protein B), and fib (encoding fibrinogen-binding protein) in all isolates of MRSA.
The detection of bap, ebpS, and fnbA was done by means of simplex PCR tests using the primers reported by Vancraeynest et al. [7].Each 50 µL PCR mixture contained 1.5 mM MgCl2, 2.5 U Taq DNA polymerase, 200 µM of each dNTP, 100 pmol of both primers, and 5 µL DNA sample.Cycling conditions for the amplification of bap included an initial denaturation step (30 seconds at 94°C) followed by 30 cycles of amplification (denaturation for 45 seconds at 94°C, annealing for 1 minute at 62°C, and elongation for 1 minute at 72°C), terminated with a 7-minute incubation step at 72°C.EbpS and fnbA were amplified using an initial denaturation step of 1 minute at 94°C followed by 30 cycles of amplification (denaturation for 45 seconds at 94°C, annealing for 45 seconds at 50°C, and elongation for 1 minute at 72°C) and a termination step of 7 minutes at 72°C.
For the detection of bbp, cna, eno and clfA, clfB, fib, and fnbB, two multiplex PCR assays were performed.Each 50 µL PCR mixture contained 3 mM MgCl2, 1.5 U Taq DNA polymerase, 40 µM of each dNTP, 100 pmol of both primers, and 5 µL DNA sample.The thermal cycling conditions for the two multiplex PCRs included an initial denaturation step (5 minutes at 94°C) followed by 25 cycles of amplification (denaturation for 1 minute at 94°C, annealing for 1 minute at 55°C, and extension for 1 minute at 72°C).The reaction was terminated with a 10-minute incubation step at 72°C.The primers and annealing temperatures used in the PCR assays are shown in Table 1.
After amplification, 8 µL of amplicon was mixed with 2 µL of loading buffer and electrophoresis was performed at 100 V for 45 minutes.After electrophoresis, gels were visualized under UV light and photographed, and the 100 bp DNA ladder was used as a DNA size marker.

Statistical analysis
Data analysis was performed using SPSS software, version 10.0 (IBM, Armonk, USA).Statistical significance was assessed using Fisher's exact test.

Detection of biofilm formation
Phenotypic production of biofilm by all strains under study was assessed using the microtiter plate test.All of the strains were biofilm positive; 21 of the 53 MRSA were high virulence, 20 strains were moderate, and 12 strains were low virulence within 24 hours.

PCR assay on MSCRAMM and biofilm genes
The PCR technique was applied to the 53 MRSA strains.The icaD gene was present in 100% of both The nucleotide sequences of ebpS (encoding elastin binding protein), eno (laminin binding protein), cna (collagen binding protein), fnbA and fnbB (fibronectin binding proteins A and B), fib (fibrinogen binding protein), clfA and clfB (clumping factors A and B), and bbp (bone sialoprotein binding protein), bap (encoding biofilm associated protein); PCR: polymerase chain reaction.
high-virulence strains and low-virulence strains, giving a 198 base pair band.All of the strains produced slime, so there was a correlation between biofilm production and presence of icaD gene in all MRSA isolates.None of the isolates harbored bap or bbp.Furthermore, 96.23% isolates were positive for fnbA, 60.37% for eno, 43.39% for clfA and clfB, 11.32% for cna, 9.34% for ebpS, 5.66% for fib, and 1.89% for fnbA (Table 2).

Discussion
The ability to form biofilm is a trait that is closely associated with bacterial persistence and virulence, and many chronic bacterial infections are now believed to be linked to the formation of biofilms [13].It has been demonstrated that strains having an ability to form biofilm additionally cause chronic polymer-associated infection [14,15].In this regard, adhesion formation is considered as one of the major virulence factors in MRSA.Providing the phenotypic and genotypic characterization of the icaD/biofilm gene may allow a better understanding of the complex process of infections caused by biofilms [16].Indeed, an increasing number of different Staphylococcus aureus adhesion molecules have been found [17].In this study, a microtiter polystyrene plate was chosen to detect biofilm formation.The presence of adherences genes should be confirmed by genotypic characterization methods.The biofilm development process requires polysaccharidic intercellular adhesin, which is synthesized by the enzymes encoded by the intercellular adhesion cluster (ica) [18].Ica expression is regulated by multiple genes such as sigB, sar, and accessory gene regulator.They may interact each other and regulate biofilm formation.
In the present study, an association was found between icaD gene detection and biofilm formation, with 100% of the strains harboring icaD and producing biofilm, which is in agreement with the findings of Liberto et al. [19].There was no difference in the distribution of the icaD gene in high-and low-virulence strains, which is in agreement with the findings of Rohde et al. [20].Our results showed high rates of biofilm formation, meaning that hospital environments may be more suitable settings for biofilm formation.Various healthcare-associated risk factors have been suggested to affect biofilm formation.The presence of ica adhesion genes may explain the role of the various adhesion mechanisms in the pathogenesis of infection.However, some studies found no association between icaD detection and biofilm formation; the presence of icaA/D genes was not always associated with in vitro formation of biofilm [7].Ruzicka et al. demonstrated that 20% of strains with ica genes did not express phenotype [21].The lack of biofilm, despite the presence of ica, could be due to several reasons, such as the inactivation of the ica operon by the action of the icaR repressor [22], or the post-transcriptional regulation [23].Biofilm is a perfect medium for the exchange of resistance plasmids [24].
None of the strains harbored the bap gene.This is in agreement with the study of Arciola et al. [25].MRSA expresses many surface proteins of the MSCRAMMs family, which specifically recognize and bind to the extracellular matrix components of the host.
The fnbA and fnbB genes contribute to the invasion and adhesion of bacteria.In the present study, a low percentage of occurrences of the fnbB gene was observed (1.89%).Taneike et al. reported that all MRSA isolated from nosocomial outbreaks in Japan lacked fnbB [26].A high occurrence of this gene (99.5%) was reported in the study of Arciola et al. [27]; this could partly be ascribed to the different technique of gene detection utilized and also to the different region of the locus analyzed by the couple of primers [28].The presence of the fnbB gene may be correlated with biofilm-forming ability [29], and appears to be a very relevant, almost essential, trait for virulence action in human hosts.
However, the gene encoding the fnbA gene was detected in 96.23% of isolates, similar to what was observed by Ikawaty et al. [28], and with other more general reports on isolates of Staphylococcus aureus from nosocomial infections.Both the clfA and clfB genes had a prevalence of 43.39%.We observed, in our study, that the strains from blood origin had a high percentage (86.95%) of both clfAB genes, which is consistent with the fact that these genes have been reported to play a determinant role in bacterial virulence [30].
Fibronectin is a high molecular weight glycoprotein, and a component of the extracellular matrix that binds to cell membrane proteins termed integrins, but can also bind to fibrin and collagen.
Fibrinogen is a glycoprotein found in blood plasma.Fibrinogen plays an important role in the blood coagulation process, in which fibrinogen is transformed into its insoluble form, fibrin.The fibrinogen-binding protein is an important virulence factor in infections caused by Staphylococcus aureus, as it not only binds to fibrinogen, but can also interfere with the aggregation of platelets and the complement cascade within the host [31][32][33].As for the fib gene, we detected it at a rate of 5.66% in our study.Elastin is the major component of elastic fibers, which are proteins that provide strength and flexibility to connective tissue [32]; we verified the presence of ebpS gene in 9.34% of our isolates.
Collagen is the most abundant group of proteins in the body and is the major component of connective tissue.The cna gene is considered an important virulence factor in staphylococcal infections [34].The incidence of cna was 11.32% in our isolates.A high occurrence of this gene was reported by Montanaro et al. (29%) [35], Nashev et al. (46.7%) [36], and Arciola et al. (46%) in Italy and Bulgaria [27], respectively.The difference in the prevalence of the cna gene in this study is probably due to different techniques in PCR detection or differences in the distribution of Staphylococcus aureus genotype in different countries.
In the present study, the bbp gene, associated with osteomyelitis and arthritis in humans [37], was absent in all strains, which is in agreement with a study by Vancraeynest et al. [38], where the bbp gene were not detected in all isolates.
In this study, 15.09% of isolates were positive for icaD and five MSCRAMM genes, which is in agreement with the findings of Tristan et al. [39], The presence of the combination of these genes, could mean that they may have a selective advantage, such as better host colonization and good genetic capacity for adherence.
However, there were no significant differences between MSCRAMM gene distribution and biofilm forming capacity (p > 0.05 for each gene comparison with exact Fisher's exact test) (Table 2).
The severity of the MRSA infections observed in the pediatric patients might be related to the high frequency of virulence factors carried by the isolates.In this study, a number of genes were associated with the development of invasive disease, contributing independently to the virulence of MRSA.Bacterial adhesion has long been considered as a virulence factor contributing to infections associated with catheters and other indwelling medical devices.In our study, 9.43% of strains were from catheters, showing a strong or moderate biofilm.The interaction of bacteria with biomaterials has been suggested to have a crucial role in conditioning the progress of these severe nosocomial infections [40].For staphylococcal species, 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 [11].
Results of a comparison between the infection types showed that the isolates from pus infections produced a strong biofilm.

Conclusions
Our findings showed the significant role of ica genes as virulence markers for staphylococcal isolates.Their association with biofilm-forming strains strongly suggests that expressions of icaD genes play a role in the pathogenic mechanisms of infections, and showed that MRSA carriage in Marrakech children was high.Most of them contained the adhesin genes.From this data, school sanitation and hygiene education programs are needed to minimize transmission of MRSA.In addition, the genetic variation of adhesion genes requires further investigation.

Table 1 .
Primers used in this study.