Molecular epidemiology, antimicrobial resistance, and virulence characteristics of predominant methicillin-resistant Staphylococcus aureus clones with strong biofilm-producing capability from a tertiary teaching hospital in China

Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent bacterial pathogens leading to various kinds of infections, but the characteristics of this superbug with both strong biofilm-producing and intracellular invasive capabilities is rarely reported. This study aimed to investigate the genotypic and phenotypic features of this superbug with above two properties.

Methods: Phenotypic resistance profiling of MRSA clinical isolates was performed via the VITEK 2 AST-GP67 Test Kit. Biofilm production was assessed via crystal violet staining and the Congo red agar (CRA) method. The biofilm-degrading activity was tested using Proteinase K, Dispersin B, and DNase I. The intracellular invasive capability was evaluated via dilution plate count and immunofluorescence assay. Genotyping was performed using multilocus sequence typing and staphylococcal protein A typing methods, and virulence genes were detected via polymerase chain reaction. Flow cytometry was performed to assess the cytotoxicity of the dominant MRSA clones.

Results: A high prevalence (21.6%) of MRSA isolates exhibiting strong biofilm-forming capability was observed in this study, including 70 strains with the highest level of biofilm production (optical density > 0.4). DNase I exhibited the most effective biofilm-degrading activity, with the biofilm-degrading percentage of 78.6% of the strains exceeding 50%. Simultaneously, 71.4% of the isolates exhibited strong invasive capability into A549 cells. ST5-t2460 (48.6%), ST59-t437 (20%), and ST239-t030 (11.4%) were identified as the predominant clones. In particular, ST5-t2460 and ST239-t030 clones exhibited broader antibiotic resistance to gentamicin, ciprofloxacin, levofloxacin, moxifloxacin, and tetracycline compared with ST59-t437 clone. In addition, a higher percentage of the isolates belonging to ST5-t2460 (91.2%) and ST239-t030 (100%) clones demonstrated stronger intracellular invasive capability relative to those belonging to ST59-t437 clone (14.3%). Furthermore, ST5-t2460 and ST239-t030 clones displayed stronger cytotoxicity and carried higher proportions of adhesion-related genes (fnbA, sdrD, sasC) and other virulence genes (sea, seb, sec, isdB, lukE-D, tsst-1).

Conclusions: This is the first report of the phenotypic-genotypic characteristics of MRSA with both strong biofilm-producing and virulence potential, with ST5-t2460, ST59-t437, and ST239-t030 clones accounting for the major genotypes. Further exploration of specific virulence genes correlating to the pathogenesis of this superbug is deemed essential for developing targeted infection control and treatment strategies in the future.

Keywords: Biofilm; Epidemiology; Infection control; MRSA; Molecular characterization; Virulence gene.

Fig. 1

Evaluation of biofilm formation capability of 1,072 clinical MRSA isolates. (A) The proportion of 1072 MRSA strains was categorized by the collection year, covering clinical samples from 2010 to 2023. (B) The proportion of biofilm-producing MRSA strains categorized by the sample source, including respiratory (throat swab, sputum, bronchoalveolar lavage fluid), blood, wound secretion, urine, and other sample types such as ascitic fluid, rectal swabs, and synovial fluid

Fig. 2

Biofilm degradation experiment of MRSA strains with strong biofilm-producing capability. (A) The proportion of strains with a biofilm degradation rate of ≥ 50% using three degrading agents among 70 MRSA strains. (B) The average degradation rates of 70 strong biofilm-producing MRSA isolates treated with single and dual degradation agents

Fig. 3

Antimicrobial susceptibility test results of 70 MRSA strains with biofilm-producing capability. (A) Abbreviations: PEN, penicillin; GEN, gentamicin; RIF, rifampicin; OXA, oxacillin; CIP, ciprofloxacin; LVX, levofloxacin; MFX, moxifloxacin; CLI, clindamycin; ERY, erythromycin; LNZ, linezolid; VAN, vancomycin; QDA, quinupristin/dalfopristin; TCY, tetracycline; TGC, tigecycline; OXSF, cefoxitin screening; DTST, clindamycin induction. (B) Distribution of 70 MRSA isolates across different specimen sources, including respiratory (throat swab, sputum, bronchoalveolar lavage fluid), blood, wound secretion, urine, and other sample types such as ascitic fluid, rectal swabs, and synovial fluid. (C) Distribution of 37 gentamicin-resistant MRSA isolates across different specimen sources

Fig. 4

Cell invasion assay of strong biofilm-producing MRSA isolates. The invasion rates of 70 MRSA isolates were determined using the dilution plate count method. The cell invasion rate of the reference strain N315 (5.03%) was used as the threshold to classify the isolates (as shown by the dashed line). Strains with an invasion rate greater than 5.03% were defined as highly invasive MRSA, while those with an invasion rate below 5.03% were classified as weakly invasive MRSA

Fig. 5

Immunofluorescence staining results. Visualization of MRSA cell invasion using immunofluorescence staining, with MRSA-760 representing a strong invasion strain (cell invasion rate = 26.47%) and MRSA-547 a weak invasion strain (cell invasion rate = 1.97%)

Fig. 6

The characteristics of phenotypic resistance and cellular invasiveness of the major MRSA clonal lineages identified in this study. (A) Among the isolates resistant to five antibiotics (GEN, gentamicin; CIP, ciprofloxacin; LVX, levofloxacin; MFX, moxifloxacin; TCY, tetracycline), the predominant sequence types were ST5, ST239, and ST59. (B) The correlation between MLST types and cell invasion ability is shown, with the y-axis indicating invasion rate (%) and the x-axis representing MLST types, where each data point reflects the invasion result of a single MRSA isolate

Fig. 7

Flow cytometry analysis of cellular virulence in three major clonal types of MRSA. Control group: living cells without bacterial treatment; ST5-458 group: strong invasive MRSA strains, cell invasion rate = 20%; ST239-144 group: strong invasive MRSA strains, cell invasion rate = 16.9%; ST59-95 group: weak invasive MRSA strains, cell invasion rate = 1.03%. Statistical significance was determined by single factor analysis of variance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001