Introduction

Staphylococcus aureus is one of the most important pathogens responsible for both nosocomial and community-acquired infections [11]. These infections, particularly those caused by methicillin-resistant Staphylococcus aureus (MRSA) strains, have been treated primarily with glycopeptide antibiotics, such as vancomycin. Unfortunately, the increased incidence of nosocomial MRSA infections and extensive use of vancomycin have led to the expansion of S. aureus strains with varying grades of resistance to vancomycin [13]. The precise mechanisms which cause reduced susceptibility to vancomycin in S. aureus isolates remain unclear. Several phenotypic, genetic and biochemical alterations can be found in vancomycin-intermediate S. aureus (VISA) strains [15]. The ability to attach to surfaces and biofilm formation is one of the most important virulence factors in S. aureus infection [6]. The development of biofilms by S. aureus is a two-step process involving an initial attachment and a subsequent maturation step. These steps are affected by several physiological factors and require step-specific factors and environmental condition [24]. In the human body, attachment to matrix proteins on the cell surface is a critical step toward S. aureus attachment and biofilm formation [20]. Although biofilm-forming properties have been well demonstrated in S. aureus, they still remain unclear among the recently emergent VISA strains. S. aureus expresses several microbial surface components recognising adhesive matrix molecules (MSCRAMMs) that interact with host extracellular ligands, such as elastin-binding protein (ebpS), laminin-binding protein (eno), collagen-binding protein (cna), fibronectin-binding proteins A and B (finbA, finbB), fibrinogen-binding protein (fib), clumping factors A and B (clfA, clfB) and bone sialoprotein-binding protein (bbp) [2]. Another significant factor required for the formation and maturation of biofilm is the polysaccharide intercellular adhesin (PIA), which is also called poly-N-acetylglucosamine (PNAG) because of its chemical composition [22]. PIA biosynthesis is catalysed by the products of the ica gene locus, which contains an N-acetylglucosamine transferase (icaA and icaD), a PIA deacetylase (icaB), a putative PIA exporter (icaC) and a regulatory gene (icaR) [10, 17, 18]. The role of biofilms in clinical infections has received increasing interest because of the detection and characterisation of genes involved in biofilm formation [8, 9]. In this study, we investigated the prevalence of MSCRAMMs and biofilm-related genes and biofilm formation abilities in VISA strains.

Materials and Methods

Bacterial Isolates

Eleven VISA isolates were chosen from a collection of 415 clinical isolates of S. aureus during 3 years (Aug 2010 to March 2013), from four university hospitals of Tehran. Isolates were confirmed as VISA by vancomycin agar screening test and agar dilution method. The minimum inhibitory concentrations (MICs) to vancomycin for all VISA isolates were between 4 and 8 μg/ml. All isolates were also reevaluated for the presence of the vanA gene by PCR. vanA gene was not found in any of the VISA strains. VISA isolates were stored at −70 °C in tripticase soy broth supplemented by 10 % glycerol before tested.

Biofilm Formation Assay

A modified microtiter plate method was followed as previously described [26]. Briefly, the wells of microtiter plate are filled with 180 µl of trypticase soy broth (TSB) containing 1 % glucose. Then, a 20 µl quantity of previously prepared bacterial suspension with turbidity equal to 0.5 Macfarland standards is added to each well. The negative control wells contain 200 µl of TSB supplemented with 1 % glucose. Incubation was carried out at 37 °C for 24 h before removal of the cultures. Then, the cells were decanted, and each well is washed three times with sterile phosphate buffered saline, fixed by methanol for 20 min, dried at room temperature and finally strained with 0.1 % saferanin. The safranin dye bound to the adherent cells was dissolved with 1 mL of 95 % ethanol per well, and the plates were read at 490 nm (A 490) using ELISA reader. Optical density cut-off (ODc) was determined. It is defined as average OD of negative control + 3 × standard deviation (SD) of negative control. Formation of biofilm by isolates was analysed and categorised relying on the absorbance of the safranin-stained attached cells. The data calculation has been shown in Table 1. Biofilm-producing S. aureus ATCC 35556 strains were used for strongly biofilm-producing control, while S. epidermidis ATCC 12228 strains were used to negative control.

Table 1 Classification of biofilm formation abilities by Mtp method
Full size table

Polymerase Chain Reaction (PCR) for icaABCD Genes

VISA isolates were selected to molecular screening for icaABCD genes using polymerase chain reaction which was carried out as previously described [10] [22]. PCR amplification was performed with an Eppendorf thermal cycler (Mastercycler® gradient). The primers, PCR condition and sizes of the expected amplification product for PCR amplification are listed in Table 2.

Table 2 List of primers used in this study
Full size table

Multiplex PCR for MSCRAMMs Genes

Nine MSCRAMM genes in different VISA isolates were examined by multiplex PCR method. Five microlitres of template DNA was added to a 50 μL reaction mixture (Amplicon. Denmark) containing 25 mM Tris/HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, all primers at various concentrations and 2.5 U Taq polymerase. Two primer groups were prepared for multiplex PCR: PCR1 to amplify bbp, cna, ebpS and eno and PCR2 to amplify fnbA, fnbB, fib, clfA and clfB [27]. The PCR products were analysed by electrophoresis in a 1.4 % agarose gel and stained with gel red. The primers, PCR condition and sizes of the expected amplification product are listed in Table 2.

Spa Typing

Determination of spa type was performed by PCR with primers forward (5′-AGACGATCCTTCGGTGAGC-3′) and reverse (5′-GCTTTTGCAATGTCATTTACTG-3′), and sequencing of polymorphic X region of spa gene was carried out as previously described [25]. The amplified spa gene fragments were purified and sequenced.

MLST

For all VISA isolates, the ST was determined by MLST. Partial gene sequences of seven housekeeping genes (arcC,aroE, glpF, gmk, pta, tpi and yqiL) were determined by PCR and direct sequencing, and their allelic profile (allele numbers) and ST were obtained using the S. aureus MLST database (http://www.mlst.net) hosted by Imperial College in London, UK [12].

Results and Discussion

Attachment to cell surfaces and inanimate surfaces, such as catheters and prosthetic devices, as well as biofilm formation are the most important processes of pathogenesis in infections caused by staphylococcal species, such as S. aureus and S. epidermidis [21]. Recently, a rapid increase in the populations of S. aureus with reduced susceptibility and resistance to vancomycin has been reported from different parts of the world [3–5, 14]. There are several reports concerning the prevalence of MSCRAMMs and biofilm-related genes from different sources of S. aureus in the literature. The study of the ability of VISA strains to adhere to surfaces and the genetic characteristics concerning MSCRAMMs and biofilm-related genes in VISA strains will improve our understanding of the correlation between reduced susceptibility to vancomycin in S. aureus and the ability of these strains to biofilm formation [2]. In the present study, all of the VISA clinical isolates examined were found to be adherent, although at different levels (Table 3). In this study, microtiter tissue culture plates were chosen to determine the ability of VISA strains to biofilm formation because using this method is easy to differentiate between weakly, moderately and strongly adherent isolates. Howden et al. analysed the biofilm-forming ability of clinical VISA strains isolated from bacteraemic patients and demonstrated reduced biofilm formation compared with vancomycin-susceptible S. aureus (VSSA) strains [16]. In another study, Sakoulas et al. analysed the biofilm-forming ability of laboratory-induced VISA strains and demonstrated enhanced biofilm formation compared with VSSA strains [23]. Our results showed that the ability of VISA strains to biofilm formation is variable because among our VISA strains, 54.5 % of isolates showed strong, 27.3 % showed moderate and 18.2 % showed weak adhesion. In contrast to previous reports, we demonstrated that the ability of biofilm formation by VISA strains is not related to the source of isolation such as laboratory-induced or clinically derived strains. These results may be caused by of the low number of VISA strains obtained in previous studies. Additionally, the significant cell wall and physiological changes occurring in VISA strains could limit or enhance biofilm formation by affecting the initial attachment or subsequent intercellular adhesion.

Table 3 Biofilm formation by microtiter tissue culture plate method
Full size table

Several reports have been published about the correlation between phenotypic methods, such as the use of microtiter tissue culture plates and the presence of ica locus and MSCRAMMs genes in S. aureus isolates from different parts of the world [2, 19]. In most of these studies, no correlation was seen between the genetic characterisation and the phenotypic biofilm assay. In a study conducted by Atshan et al. that was similar to our study, there was no direct correlation between morphology and the distribution of MSCRAMM genes [2]. Thus, the disagreement between phenotypic and genotypic characterisation may be due to variation and heterogeneity in the genetic backgrounds. In addition, biofilm formation is affected by a variety of factors such as environmental conditions [1]. The nine MSCRAMMs and four biofilm-related genes were examined in VISA and other strains using a PCR method. The clfaA, clfB, fnbA, icaD and fib genes were detected in all of the mentioned isolates. However, the bbp gene was not detected in any of the VISA strains. Other MSCRAMMs had varying prevalence among the VISA strains: the prevalence of eno and cna was 90.1 %, whereas the prevalences of ebpS, bbp and fnbB in VISA strains were 18.2, 0 and 54.5 %, respectively. The prevalences of icaA, icaB, icaC and icaD in VISA strains were 63.7, 63.7, 90.1 and 100 %, respectively (Table 4). No relationship was observed between the source of bacteria, such as wards and type of specimens and the potential for biofilm formation among VISA strains. No direct correlation between the distribution of adhesin genes and biofilm formation was seen. However, VISA strains with the same frequency of MSCRAMMs and biofilm-related genes give different results in phenotypic tests. All of the examined VISA strains harboured at least four MSCRAMM genes including clfA, clfA, fnbA and fib. According to Howden et al., the variation in the prevalence of MSCRAMM genes depends on the several factors, such as species and source of isolation [15]. There was no direct relationship between the distribution of MSCRAMMs and biofilm-related genes and the ability to biofilm formation, which indicates that a single gene or a subset of biofilm-related genes cannot be employed as an indicator of the ability to biofilm formation.

Table 4 Relationships among the presence of MSCRAMMs and biofilm-related genes, adherence capacity, ST and Spa types in 11 clinical isolates of S. aureus isolates naming VISA
Full size table

Spa typing of the 11 isolates revealed six different spa types. Repeats among the spa types varied from 2 (t2467) to 12 (t12925). A dendrogram was constructed for all eleven isolates in order to visualise the relationships among the spa types (Fig. 1). Among the spa types, t030 was the most prevalent and was detected in 4 (36.4 %) strains. In addition, among the six spa types, one new spa type was identified and deposited in the spa database. Isolates that belong to same Spa and ST types showed similar adherence capacity in the Mtp assay, but significant differences were observed between different Spa types. For example, all isolates belonging to spa type t030 were found to form a strongly adherent biofilm, whereas all isolates belonging to spa type t037 were observed to have weak biofilm formation using the microtiter plate method. This is in agreement with the findings of Croes et al., who reported differences in biofilm formation among S. aureus isolates related to the genetic background of protein A (Spa) [7].

Fig. 1
figure 1

Dendrogram comparing spa types and biofilm formation ability of VISA isolates

Full size image

In summary, the findings of this study, which used both phenotypic and genotypic methods, demonstrated that genotypically different VISA isolates have different abilities to produce biofilms. This diversity in biofilm characteristics is because of the differences in genetic background, such as Spa type. It has been shown that VISA strains have different capacities to adhere to surfaces and biofilm formation. This may be caused by several factors, such as physiological and structural conditions, and not because of their differences in the presence of MSCRAMMs or biofilm-related genes.