Summary

Introduction: The biofilm formation ability plays an important role in the pathogenesis of Staphylococcus aureus infections. This study aimed to investigate the biofilm production ability of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates and evaluate the relationship between their antimicrobial resistance profile and biofilm formation ability.
Materials and Methods: A total of 50 MRSA and 50 MSSA isolates were examined. The antimicrobial susceptibility testing of isolates was performed using the disk diffusion method. The broth microdilution method was used to determine the minimum inhibitor concentrations (MICs) of vancomycin and teicoplanin. The biofilm formation ability of isolates was tested on Congo Red Agar. The presence of icaA, icaD, IS256, and eno genes was investigated by polymerase chain reaction.
Results: Both MRSA and MSSA isolates were found susceptible to vancomycin, teicoplanin, chloramphenicol, and linezolid. Two MRSA and 2 MSSA isolates were determined as heterogeneous vancomycin-intermediate S. aureus. No significant difference was observed between the biofilm formation ability of MRSA and MSSA isolates. The eno and icaD genes were detected in 100% of both MSSA and MRSA isolates. The icaA gen was detected in all MRSA and 49 MSSA isolates. The IS256 was detected in 35 of the 50 MRSA isolates. None of the MSSA isolates were positive for the IS256. The amikacin, gentamicin, ciprofloxacin, levofloxacin, rifampin, clindamycin, and tetracycline resistance rates in IS256- positive MRSA isolates were significantly higher than those IS256-negative MRSA isolates. The mean MIC values of vancomycin and teicoplanin in IS256-positive MRSA isolates were significantly higher than those in IS256-negative MRSA isolates.
Conclusion: This study revealed that the presence of the IS256 sequence was correlated with antimicrobial resistance, especially MRSA isolates.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is accepted as the most common resistant pathogen that leads to both community and hospital-acquired infections worldwide. Most MRSA isolates are not only resistant to beta-lactams, but also other antimicrobial agents, such as macrolides, tetracyclines, fluoroquinolones, aminoglycosides, etc^[1]. Limited options are available for the treatment of infections caused by these resistant isolates. The glycopeptide antibiotics, especially vancomycin, were used for the treatment of serious infections caused by MRSA isolates^[2].

S. aureus strains with vancomycin minimum inhibitory concentration (MIC) of >2 μg/ml is accepted as non-susceptible according to the European Committee on Antimicrobial Sensitivity Testing (EUCAST) criteria^[3]. The Clinical Laboratory Standards Institute (CLSI) guidelines define vancomycin breakpoints for S. aureus strains as follows: susceptible at a vancomycin MIC of 2 μg/ml, intermediate susceptible at 4-8 μg/ml, and resistant at 16 μg/ml^[4]. A recent global report has documented the prevalence of vancomycin-resistant S. aureus (VRSA), vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA) has been significantly increasing worldwide^[5]. The hVISA phenotype contains VISA subpopulations with high levels of vancomycin MICs. An infection caused by hVISA is usually correlated with vancomycin treatment failure^[2].

Moreover, another reason for the treatment failure in MRSA infections is the biofilm formation ability of MRSA isolates. Biofilm plays an important role in the pathogenesis of MRSA infections. The biofilm formation ability of S. aureus isolates is correlated with many serious chronic infections, such as osteomyelitis, urinary tract infections, catheter-related infections, and endocarditis. Methicillin-resistant Staphylococcus aureus isolates can colonize and produce biofilm matrix on implanted medical devices. This biofilm is mainly composed of a specific polysaccharide antigen called polysaccharide intercellular antigen (PIA). The icaADBC (intercellular adhesionADBC) operon in S. aureus isolates is responsible for PIA synthesis^[6]. The icaADBC operon contains icaA, icaD, icaB, and icaC genes. Thus, the presence of these genes in S. aureus isolates is directly linked with the biofilm formation ability. The eno gene encodes the a-enolase in S. aureus isolates. This enzyme is responsible for laminin-binding and biofilm formation activation^[7]. The presence of insertion sequence element IS256 has been also associated with biofilm formation in staphylococci^[8]. The insertion or excision of IS256 into the intercellular adhesion (ica) gene locus changes the biofilm phases of S. aureus^[9]. The biofilm matrix supplies a physical barrier against the entrance of the antimicrobial agents and the host immune system^[10]. Infection associated with a biofilm that is formed by MRSA is an important medical problem because of its limited therapeutic strategies^[6].

This study aimed to investigate the biofilm production ability of MRSA and MSSA isolates and evaluate the relationship between their antimicrobial resistance profile and biofilm formation ability.

Methods

MRSA and MSSA Isolates

A total of 50 MRSA and 50 MSSA isolates were included in this study. All isolates were randomly selected from the culture collection of the Bacteriology Laboratory of our hospital. The MRSA and MSSA isolate distribution by clinical specimens are shown in Table 1. The identification of isolates was conducted using the automated VITEK2^® system (bioMérieux, France). Gram staining, catalase, coagulase, and the production of DNase were simultaneously tested to confirm the identity of isolates.

Table 1: The distribution of MRSA and MSSA isolates by clinical specimens

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing to a penicillin (1 IU), tetracycline (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), linezolid (10 µg), erythromycin (15 µg), rifampin (5 µg), gentamicin (10 µg), amikacin (30 µg), chloramphenicol (30 µg), clindamycin (2 µg), ceftaroline (5 µg), and trimethoprim-sulfamethoxazole (1.25/23.75 μg) (Bioanalyse, Turkey) was performed using Kirby-Bauer disk diffusion method according to the EUCAST criteria^[3]. Methicillin resistance was determined with cefoxitin disk (30 μg) (Bioanalyse, Turkey) following the EUCAST (2019) guidelines^[3]. Inducible and constitutive macrolide-lincosamide-streptogramin B (iMLS_B and cMLS_B) resistance was tested using the double-disk test.

The reference broth microdilution method was used to determine the MIC of vancomycin and teicoplanin. Vancomycin hydrochloride (Sigma-Aldrich, USA) and teicoplanin (Carbosynth Ltd, UK) was dissolved in sterile water and used to prepare stock solutions. Both vancomycin and teicoplanin susceptibilities were tested at concentrations from 0.125 µg/mL to 32 µg/ml. All samples were tested in duplicate. The EUCAST criteria were used to interpret the test results^[3]. S. aureus ATCC 29213 was used as a quality control strain.

Teicoplanin Agar Screening Method

All MSSA and MRSA isolates were screened for hVISA. Mueller Hinton agar (Conda, Spain) plates containing 5 mg/L teicoplanins (Carbosynth Ltd, UK) were prepared and used according to the EUCAST recommendations to detect hVISA^[3]. After inoculating 10 μl of bacterial suspension adjusted to 2 McFarland on the agar plate surfaces, plates were incubated at 35 °C for 48 h and read. S. aureus ATCC 700698 (Mu3) (hVISA), S. aureus ATCC 700699 (Mu50) (VISA), and S. aureus ATCC 29213 (vancomycin susceptible S. aureus) were used as control strains.

Modified Population Analysis Profile-area Under the Curve Method

The population analysis profile-area under the curve (PAP-AUC) was performed as previously described^[11]. The bacterial suspension was inoculated onto Brain-Heart Infusion agar plates (Conda, Spain) containing 0, 0.5, 1, 2, 4, and 8 μg/ml of vancomycin. After 24 h of incubation at 35 °C, colony growth was counted as log₁₀ CFU/ml. The logarithmic count of each isolate was plotted against the vancomycin concentrations on the graph. This graph was used to calculate the AUC. The AUC ratio was calculated by dividing the AUC of the test strain by the AUC of the reference Mu3 strain. The PAP-AUC ratio was interpreted as follows: 0.90 as susceptible, 0.90-1.3 as hVISA, and 1.3 as VISA^[11].

Phenotypic Analysis of Biofilm Formation

The phenotypic analysis of biofilm formation of the MSSA and MRSA isolates was tested on Congo Red Agar (CRA) medium as previously described^[12]. The CRA medium was prepared by adding sucrose (Isolab, Turkey) (50 g/L), agar (10 g/L), and Congo Red stain (CDH, India) (0.8 g/L) to the brain-heart infusion broth (Conda, Spain) (37 g/L). After 48 h of incubation at 37 °C, bacterial colony morphology was evaluated. Black colonies on CRA medium were defined as biofilm producer, pink as non-producer, and burgundy as borderline^[13]. S. epidermidis ATCC 35984 and S. epidermidis ATCC 12228 strains were used as positive and negative controls, respectively.

Detection of icaA, icaD, IS256, and eno Genes

Genomic DNA was extracted using the high pure polymerase chain reaction (PCR) template preparation kit (Roche, Germany) according to the manufacturer’s recommendations. The primer sets described by Vancraeynest et al.^[14] and Montanaro et al.^[15] were used for the amplification of eno and IS256 genes, respectively. The multiplex PCR method was used to detect the eno and IS256 genes. The initial step (94 °C for 3 min) was followed by 35 cycles with denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, elongation at 72 °C for 1 min, and a final step at 72 °C for 10 min. The presence of eno and IS256 gene regions was confirmed in our fiftieth MRSA isolate by sequencing analysis. Then we used this strain as a positive control.

The molecular detection of icaA and icaD genes was performed as previously described by Vasudevan et al.^[16] with slight modifications. PCR conditions were the following: an initial temperature of 94 °C for 3 min, followed by 35 cycles with denaturation at 94 °C for 30 sec, annealing at 57.8 °C for 30 sec, elongation at 72 °C for 1 min, and a final step at 72 °C for 10 min. The fifth MRSA isolates in our study confirmed the existence of icaA and icaD gene regions by sequencing analysis and used as a positive control. S. epidermidis ATCC 12228 was used as a negative control.

After the amplification, the amplicons were run on a 1.5% agarose gel (GeneOn, Germany), containing 0.5 μg/ml ethidium bromide (Sigma-Aldrich, USA) at 80 V for 2.5 h and visualized using a transilluminator (Sigma-Aldrich, USA).

Statistical Analysis

All statistical analyses were performed using GraphPad Prism 9 demo version statistical software. The association between the categorical variables was determined using the chi-square (χ²) test and Student’s t-test. P values of <0.05 were considered significant.

Results

Antimicrobial Drug Resistance Profiles

All isolates were found susceptible to vancomycin, teicoplanin, chloramphenicol, and linezolid. The antimicrobial resistance rates of penicillin, tetracycline, ciprofloxacin, levofloxacin, linezolid, erythromycin, rifampin, gentamicin, amikacin, chloramphenicol, clindamycin, ceftaroline, and trimethoprim-sulfamethoxazole against MRSA and MSSA isolates are shown in Table 2. The significant differences in antimicrobial-resistant rates to penicillin, tetracycline, ciprofloxacin, levofloxacin, erythromycin, rifampin, gentamicin, amikacin, clindamycin, and ceftaroline were detected between MRSA and MSSA isolates. iMLSB and cMLSB were detected in 29% and 3% of all isolates, respectively. The MIC ranges, MIC₅₀, and MIC₉₀ values of vancomycin and teicoplanin for MRSA and MSSA isolates are shown in Table 3. All tested MRSA and MSSA isolates were found to be susceptible to vancomycin and teicoplanin.

Table 2: The antimicrobial resistance rates of penicillin, tetracycline, ciprofloxacin, levofloxacin, linezolid, erythromycin, rifampicin, gentamicin, amikacin, chloramphenicol, clindamycin, ceftaroline, and trimethoprim-sulfamethoxazole against MRSA and MSSA isolates

Table 3: The MIC ranges, MIC₅₀, and MIC₁₀₀ values of vancomycin and teicoplanin

Detection of hVISA

Eleven MRSA and seven MSSA isolates were found to be hVISA according to the teicoplanin agar screening method. The PAP-AUC analysis was performed to confirm the teicoplanin agar screening method results. The PAP-AUC analysis confirmed 2 of 11 MRSA and 2 of 7 MSSA isolates as hVISA. Among the 4 hVISA isolates, 3 (1 MRSA and 2 MSSA) were isolated from the wound, whereas 1 (1 MRSA) from the sputum.

Production of Biofilm

All MRSA and 48 MSSA isolates were determined as biofilm producers on the CRA medium. No significant difference was observed between the biofilm formation ability of MRSA and MSSA isolates (p>0.05). Non-biofilm producer 2 MSSA isolates were found to be susceptible to all antimicrobials except penicillin. Among the biofilm producer isolates, 1 of the MRSA and 4 of the MSSA isolates was defined as borderline phenotypically. Two of the 4 borderline biofilm producer MSSA isolates were susceptible to all tested antimicrobial agents, whereas the other 2 isolates were resistant only to penicillin.

Detection of icaA, icaD, IS256, and eno Genes

The eno and icaD genes were detected in 100% of both MSSA and MRSA isolates. The icaA gen was detected in all MRSA and 49 MSSA isolates. The agarose gel electrophoresis of icaA and icaD genes of MRSA isolates is shown in Figure 1.

The prevalence of the eno, icaD, icaA, and IS256 genes among the biofilm producer S. aureus isolates were 100%, 100%, 99%, and 36%, respectively (Figures 1, 2).

Figure 1: Agarose gel-electrophoresis of icaA and icaD genes of MRSA isolates
M: 100 bp DNA ladder, PC: Positive control, NC: Negative control

Figure 2: Agarose gel-electrophoresis of eno and IS256 genes of MRSA isolates
M: 100 bp DNA ladder, PC: Positive control, NC: Negative control

The eno, icaD, and icaA genes were observed in 2 of the non-biofilm producer MSSA isolates. The IS256 was detected in 35 of the 50 MRSA isolates. None of the MSSA isolates were positive for IS256 (Figure 2). All IS256-positive MRSA isolates were also identified as biofilm producers. The resistance rates to penicillin, tetracycline, ciprofloxacin, levofloxacin, erythromycin, rifampin, gentamicin, amikacin, clindamycin, and ceftaroline of IS256-positive S. aureus isolates were significantly higher than those IS256 negative S. aureus isolates (p<0.02, p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.0001, and p<0.0002, respectively) (Table 4). Additionally, the amikacin, gentamicin, ciprofloxacin, levofloxacin, rifampin, clindamycin, and tetracycline resistance rates in IS256-positive MRSA isolates were significantly higher than those IS256-negative MRSA isolates (p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.002, and p<0.0005, respectively) (Table 5). The mean MIC values of vancomycin and teicoplanin in IS256-positive S. aureus isolates were significantly higher than those in IS256-negative S. aureus isolates (p<0.0001, p<0.0001, respectively) (Table 6).

Table 4: The antimicrobial resistance profiles of IS256 (+) and IS256 (−) S. aureus isolates

Table 5: The antimicrobial resistance profiles of IS256 (+) and IS256 (−) MRSA isolates

Table 6: Vancomycin and teicoplanin mean MICs of IS256 (+) and IS256 (−) isolates

The mean MIC values of vancomycin and teicoplanin in IS256-positive MRSA isolates were significantly higher than those in IS256- negative MRSA isolates (p<0.002, p<0.0001, respectively) (Table 6).

Discussion

This study revealed that none of the S. aureus isolates were resistant to vancomycin, teicoplanin, chloramphenicol, and linezolid. All MSSA isolates were also susceptible to ceftaroline and rifampin. Methicillin-resistant Staphylococcus aureus isolates were more resistant to tested antimicrobial agents except for trimethoprim-sulfamethoxazole. Similar results were reported by previous researchers^{[1, 17-19]}.

The results of the broth microdilution method yielded that the vancomycin MIC range was 0.5-1 μg/ml in all S. aureus isolates. The SENTRY Antimicrobial Surveillance Program has examined antimicrobial susceptibility of 191,460 clinical S. aureus isolates between 1997 and 2016 and revealed only 1 S. aureus isolates showing the MIC value of 8 μg/ml among all the isolates^[1]. According to the teicoplanin agar screening method results, 11 of the 50 MRSA and 7 of the 50 MSSA isolates were detected as suspicious hVISA isolates; however, only 4 (2 of the 11 MRSA and 2 of the 7 MSSA) (4%) were hVISA according to modified PAP-AUC reference method. The prevalence of hVISA varies with geographical region. Results from the recent meta-analysis research have indicated that the prevalence of hVISA was 4.7% in Asia, 4.4% in Europe, and 5.2% in America. The same report also documented that the prevalence of hVISA has been increasing, especially in Asia and America^[5].

PAP-AUC method is accepted as a gold standard method for detecting hVISA strains; however, it is a laborious procedure for routine application. The EUCAST has recommended a macro gradient test, glycopeptide resistance detection gradient test, and teicoplanin agar screening method for hVISA screening^[3]. Alternatively, for hVISA screening, brain-heart infusion agar supplemented with 6 µg/ml of vancomycin (BHIA6V) has been recommended by the CLSI^[4]. Wootton et al.^[20] have revealed the teicoplanin agar screening method and macro gradient test had high sensitivity and specificity compared with BHIA6V, which compared the screening methods for hVISA. They also emphasized that the teicoplanin agar screening method was relatively low in cost compared with the macro gradient test. Therefore, we preferred the teicoplanin agar screening method for hVISA screening in our study. However, the percentage of false-positive results in our study was higher than the study by Wootton et al.^[20]. Additionally, our study noticed that 2 of the 4 hVISA strain were MSSA. Previous studies on hVISA were mainly focused on MRSA strains although decreased sensitivity to glycopeptides was also observed in MSSA isolates. Hu et al.^[21] revealed that 10% and 0.5% MSSA isolates were hVISA and VISA, respectively. Therefore, we have suggested that MSSA isolates must be tested for glycopeptide susceptibility to determine the true prevalence and revise the treatment strategies.

In this study, 98% of S. aureus isolates have been identified as biofilm producers. Similar results were reported by previous researchers^{[22, 23]}. Among the S. aureus isolates, only 2 of the MSSA isolates were detected as non-biofilm producers. These 2 MSSA isolates were resistant to only penicillin among the tested antimicrobial agents. No significant difference was detected between the methicillin resistance and biofilm formation ability in S. aureus isolates (p>0.05). This result is in concordance with the findings reported by Smith et al.^[24]. Contrary to our results, higher rates of multidrug resistance and methicillin resistance among the biofilm producer S. aureus isolates were reported by Belbase et al.^[25].

In the present study, the genes responsible for biofilm production were analyzed using the PCR method. The eno and icaD genes were found in all MRSA and MSSA isolates. The eno gene encodes the laminin-binding protein responsible for the S. aureus adherence to the extracellular matrix^[7]. This matrix is mainly composed of PIA. The PIA is produced by the N-acetylglucosaminyltranferase enzyme synthesized by the expression of the icaADBC operon, especially the icaA gene. Co-expression of icaA and icaD genes causes a substantially increased activity of acetylglucosaminyltranferase^[26]. Our study detected icaD and icaA genes in 2 of the non-biofilm producer MSSA isolates. This may be explained by insufficient phenotypic methods to identify biofilm producer isolates or different levels of expression of the genes required for biofilm production. Beloin et al.^[27] revealed that the nature of the isolates also plays role in the expression levels.

Kwon et al.^[28] revealed that the prevalence of IS256 (insertion sequence) was correlated with biofilm formation. In our study, IS256 was present in 36% of the biofilm producer S. aureus isolates. All IS256-positive S. aureus isolates were methicillin-resistant. The increased resistance rates were detected for penicillin, tetracycline, ciprofloxacin, levofloxacin, erythromycin, rifampin, gentamicin, amikacin, clindamycin, and ceftaroline in IS256-positive S. aureus isolates. Kwon et al.^[28] documented that the prevalence of IS256 was associated with multidrug resistance in S. aureus. Lyon et al.^[29] revealed that IS256 had an important role in increasing aminoglycoside resistance in S. aureus isolates. Our study revealed higher antimicrobial resistance rates among IS256-positive MRSA isolates compared with IS256-negative MRSA isolates, except for penicillin and erythromycin. Additionally, we observed the mean MICs of vancomycin and teicoplanin for IS256-positive MRSA isolates as significantly higher than those for IS256- negative isolates. Maki et al.^[30] demonstrated that the insertion of IS256 into the tcaA gene region has caused the teicoplanin resistance in S. aureus.

In this study, the IS256 sequence was not detected in any of the MSSA isolates. Our results were in concordance with Kwon et al.^[28]. They have suggested that the transposition or rearrangement of IS256 in the chromosome may contribute to the methicillin resistance of S. aureus^[28].

The main limitation of this study was the low sampling size. Another limitation was the limited number of the tested biofilm-related genes.

Conclusion

All MRSA and MSSA isolates were found susceptible to vancomycin, teicoplanin, chloramphenicol, and linezolid. Two MRSA and two MSSA isolates were determined as hVISA. No significant difference was observed between the biofilm formation ability of MRSA and MSSA isolates. The eno and icaD genes were detected in all isolates. Our results revealed that the presence of the IS256 sequence was correlated with antimicrobial resistance, especially methicillin resistance in S. aureus isolates.

Ethics

Ethics Committee Approval and Informed Consent: Our study is an in vitro study it does not require ethics committee approval and patient consent.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Laboratory and Medical Practices: B.Ç.T.D., G.Y., B.O., E.S.T., Concept: B.Ç.T.D., G.Y., B.O., Y.B., Design: G.Y., B.O., Y.B., Data Collection or Processing: B.Ç.T.D., G.Y., E.S.T., A.Y., Analysis or Interpretation: A.Y., Literature Search: B.Ç.T.D., G.Y., Writing: G.Y., A.Y.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This study was supported by the Tokat Gaziosmanpaşa University Research Fund, project no: 2020/51.