A cross-sectional molecular epidemiological study of biofilm-producing methicillin-resistant Staphylococcus aureus

Abstract

There is growing concern regarding biofilm-producing methicillin-resistant Staphylococcus aureus (MRSA) due to the sudden rise in infection rates and associated morbidity and mortality. Therefore, epidemiological studies, including molecular typing and correlation analysis, are essential for understanding this pathogen. This cross-sectional study investigated epidemiological factors and correlations in MRSA isolates. A total of 300 clinical samples were collected between January and March 2023 from 2 healthcare facilities, including various sample types such as sputum, blood, urine, pus, wound swabs, and other body fluids. This study employed various phenotypic and genotypic methodologies, including adherence assays using standard microtiter plates, the Congo red agar method, antimicrobial resistance and virulence profiling, and multi-locus sequence typing. Among 300 clinical samples from 2 healthcare facilities in Egypt, 94 MRSA isolates were confirmed as biofilm producers. Phylogenetic analysis revealed 8 distinct sequence types (ST8, ST80, ST239, ST15, ST22, ST113, ST398, ST984), found in surgical unit samples across both facilities. Notably, ST22-MRSA was present in all departments, indicating its widespread nature and potential for cross-departmental transmission. ST239-MRSA, the most prevalent strain (22.3%), was found in all departments except burn units. Alarmingly, 95.7% of isolates exhibited multidrug-resistant patterns. However, resistance to vancomycin and imipenem was low among biofilm-producing isolates. The high diversity of MRSA strains suggests multiple sources of infection rather than a single origin. Although most isolates were unrelated, the presence of 2 ST80 isolates in sputum samples from the same unit underscores the importance of targeted infection control within and between hospital areas. ST8-MRSA strains carrying the vanA gene were predominantly identified in body fluid samples, highlighting the need for regular testing in such cases. The diversity of MRSA strains across hospital departments indicates a complex infection landscape with no single source. Although certain genetic markers are linked to specific sequence types, they are not reliable indicators of MRSA clonality. These findings emphasize the need for strict infection control measures and regular testing, particularly for ST8-MRSA in body fluids.

Keywords: MRSA; biofilm; clonality; diversity; sequence types.

Figure 1.

A fan dendrogram of biofilm producing MRSA Strains Based on MLST. A fan dendrogram was constructed using multilocus sequence typing (MLST) data to illustrate the phylogenetic relationships among the methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in this study. The standard strains included in the analysis were ST8 (NCTC 8325), ST15 (NCTC 12491), ST22 (NCTC 10442), ST239 (NCTC 12493), ST80 (NCTC 12491), ST984 (NCTC 13459), ST113 (NCTC 13350) and ST398 (NCRC 10588). ST8 clade represented in purple, ST15 clade was highlighted in deep green. The light-yellow section of the dendrogram contains the ST22, the light green segment of the dendrogram shows the ST398, other sequence types like ST80, ST113, and ST239 represented in bluse, light and deep pink. MLST = multilocus sequence typing, MRSA = methicillin-resistant Staphylococcus aureus.

Figure 2.

Heatmap of biofilm producing MRSA isolates based on antimicrobial agents, resistance genes, and virulence genes. This comprehensive heatmap illustrates the complex relationship between the MRSA isolates’ antimicrobial resistance profiles, the presence of specific resistance and virulence genes, the severity of the infections, the timing of sample collection, the healthcare facilities involved, the departments from which samples were collected, and the types of samples analyzed. Each row represents a unique isolate including ST239 = biofilm producing MRSA sequence types 239, ST8 = biofilm producing MRSA sequence types 8, ST15 = biofilm producing MRSA sequence types 15, ST22 = biofilm producing MRSA sequence types 22, ST80 = biofilm producing MRSA sequence types 80, ST113 = biofilm producing MRSA sequence types 113, ST398 = biofilm producing MRSA sequence types 398, ST984 = biofilm producing MRSA sequence types 984, while the columns represent different criteria, including C = chloramphenicol, OX = oxacillin, CRO = ceftriaxone, FOX = cefoxitin, RIF = rifampicin SV, TE = tetracycline, VA = vancomycin, DA = clindamycin, SXT = trimethoprim‐sulfamethoxazole, E = erythromycin, IPM = imipenem, CIP = ciprofloxacin, CN = gentamicin, lukS = panton-valentin leukocidin, tst = toxic shock syndrome toxin-1, eta = exfoliative toxins A, etb = exfoliative toxins B, sea = Staphylococcal enterotoxins A, seb = Staphylococcal enterotoxins B, sec = Staphylococcal enterotoxins C, sed = Staphylococcal enterotoxins D, see = Staphylococcal enterotoxins E, seg = Staphylococcal enterotoxins G, seh = Staphylococcal enterotoxins H, sei = Staphylococcal enterotoxins I, sej = Staphylococcal enterotoxins J, clfA = clumping factors A gene, hla = alpha hemolysin proteins, vanA = vancomycin resistance gene A, vanB = vancomycin resistance gene B, ermE = erythromycin resistance gene, tetK = tetracycline resistance gene, B (blood, 1:14), S (sputum, 1:16), F (body fluid, 1:7), U (urine, 1:22), P (pus, 1:25), and W (wound swab, 1:10), HC1 = healthcare facilities 1, HC2 = healthcare facilities 2.

Figure 3.

Circular dendrogram of biofilm producing MRSA within each sequence types and clinical samples. (A) Fan dendrogram showing the relatedness of biofilm producing MRSA within each sequence types, (B) fan dendrogram showing the relatedness of biofilm producing MRSA within each clinical samples, the dendrogram effectively groups the MRSA sequence types based on their genetic similarities, with a focus on their resistance patterns. Each variable (lan1 to lan29) represents a specific genetic marker or resistance gene, and the color coding provides a visual representation of whether each sequence type is resistant or sensitive to the variable in question. ST239 = biofilm producing MRSA sequence types 239 (a:u), ST8 = biofilm producing MRSA sequence types 8 (a:k), ST15 = biofilm producing MRSA sequence types 15 (a:j), ST22 = biofilm producing MRSA sequence types 22 (a:i), ST80 = biofilm producing MRSA sequence types 80 (a:p), ST113 = biofilm producing MRSA sequence types 113 (a:i), ST398 = biofilm producing MRSA sequence types 398 (a:j), ST984 = biofilm producing MRSA sequence types 984 (a:e). For panel A lan1:lukS, lan2:sea, lan3:see, lan4:sec, lan5:seb, lan6:sed, lan7: clfA, lan8:hla, lan9:tst, lan10:eta, lan11:etb, lan12:OX, lan13:VA, lan14:FOX, lan15:CRO, lan16:C, lan17:SXT, lan18:CN, lan19:E, lan20:DA, lan21:CIP, lan22:RIF, lan23:TE, lan24:IPM, lan25:vanA, lan26:vanB, lan27:tetK, lan28:ermE. For panel B lan1: lukS, lan2:sea, lan3:see, lan4:sec, lan5:seb, lan6:sed, lan7:clfA, lan8:tst, lan9:eta, lan10:etb, lan11:VA, lan12:CRO, lan13:C, lan14:SXT, lan15:CN, lan16:E, lan17:DA, lan18:CIP, lan19:RIF, lan20:TE, lan21:IPM, lan22:vanA, lan23:vanB, lan24:ermE, lan25:tetK, lan26:ST239, lan27:ST8, lan28:ST15, lan29:ST22, lan30:ST80, lan31:ST113, lan32:ST398, lan33: ST984. B (blood, 1:14), S (sputum, 1:16), F (body fluid, 1:7), U (urine, 1:22), P (pus, 1:25), and W (wound swab, 1:10). The red color indicates the resistance pattern or the presence of this variable, while blue color indicates the sensitivity pattern or the absence of this variable. The arrow indicates the 2 related isolates, which share identical antimicrobial resistance and virulence patterns.

Figure 4.

Pairwise correlation (r) among all investigated biofilm producing MRSA regarding to the sequence types. ST239 = biofilm producing MRSA sequence types 239, ST8 = biofilm producing MRSA sequence types 8, ST15 = biofilm producing MRSA sequence types 15, ST22 = biofilm producing MRSA sequence types 22, ST80 = biofilm producing MRSA sequence types 80, ST113 = biofilm producing MRSA sequence types 113, ST398 = biofilm producing MRSA sequence types 398, ST984 = biofilm producing MRSA sequence types 984, C = chloramphenicol, OX = oxacillin, CRO = ceftriaxone, FOX = cefoxitin, RIF = rifampicin SV, TE = tetracycline, VA = vancomycin, DA = clindamycin, SXT = trimethoprim‐sulfamethoxazole, E = erythromycin, IPM = imipenem, CIP = ciprofloxacin, CN = gentamicin, lukS = panton-valentin leukocidin, tst = toxic shock syndrome toxin-1, eta = exfoliative toxins A etb = exfoliative toxins B, sea = Staphylococcal enterotoxins A, seb = Staphylococcal enterotoxins B, sec = Staphylococcal enterotoxins C, sed = Staphylococcal enterotoxins D, see = Staphylococcal enterotoxins E, seg = Staphylococcal enterotoxins G, seh = Staphylococcal enterotoxins H, sei = Staphylococcal enterotoxins I, sej = Staphylococcal enterotoxins J, clfA = clumping factors A gene, hla = alpha hemolysin proteins, vanA = vancomycin resistance gene A, vanB = vancomycin resistance gene B, ermE = erythromycin resistance gene, tetK = tetracycline resistance gene, B = blood, S = sputum, F = body fluid, U = urine, P = pus, W = wound swab, HC1 = healthcare facilities 1, HC2 = healthcare facilities 2.

Figure 5.

Pairwise correlation (r) among biofilm producing MRSA isolated in healthcare facility 1 regarding to the sample types, severity, time of collection, hospital departments. C = chloramphenicol, OX = oxacillin, CRO = ceftriaxone, FOX = cefoxitin, RIF = rifampicin SV, TE = tetracycline, VA = vancomycin, DA = clindamycin, SXT = trimethoprim‐sulfamethoxazole, E = erythromycin, IPM = imipenem, CIP = ciprofloxacin, CN = gentamicin, lukS = panton-valentin leukocidin, tst = toxic shock syndrome toxin-1, eta = exfoliative toxins A etb = exfoliative toxins B, sea = Staphylococcal enterotoxins A, seb = Staphylococcal enterotoxins B, sec = Staphylococcal enterotoxins C, sed = Staphylococcal enterotoxins D, see = Staphylococcal enterotoxins E, seg = Staphylococcal enterotoxins G, seh = Staphylococcal enterotoxins H, sei = Staphylococcal enterotoxins I, sej = Staphylococcal enterotoxins J, clfA = clumping factors A gene, hla = alpha hemolysin proteins, vanA = vancomycin resistance gene A, vanB = vancomycin resistance gene B, ermE = erythromycin resistance gene, tetK = tetracycline resistance gene, ICU = intensive care unit. Red and blue squares represent positive and negative correlations, respectively. The color legend corresponds to the correlation coefficient (r), with darker shades indicating stronger positive or negative correlations.

Figure 6.

Pairwise correlation (r) among biofilm producing MRSA isolated in healthcare facility 2 regarding to the sample types, severity, time of collection, hospital departments. C = chloramphenicol, OX = oxacillin, CRO = ceftriaxone, FOX = cefoxitin, RIF = rifampicin SV, TE = tetracycline, VA = vancomycin, DA = clindamycin, SXT = trimethoprim‐sulfamethoxazole, E = erythromycin, IPM = imipenem, CIP = ciprofloxacin, CN = gentamicin, lukS = panton-valentin leukocidin, tst = toxic shock syndrome toxin-1, eta = exfoliative toxins A etb = exfoliative toxins B, sea = Staphylococcal enterotoxins A, seb = Staphylococcal enterotoxins B, sec = Staphylococcal enterotoxins C, sed = Staphylococcal enterotoxins D, see = Staphylococcal enterotoxins E, seg = Staphylococcal enterotoxins G, seh = Staphylococcal enterotoxins H, sei = Staphylococcal enterotoxins I, sej = Staphylococcal enterotoxins J, clfA = clumping factors A gene, hla = alpha hemolysin proteins, vanA = vancomycin resistance gene A, vanB = vancomycin resistance gene B, ermE = erythromycin resistance gene, tetK = tetracycline resistance gene, ICU = intensive care unit. Red and blue squares represent positive and negative correlations, respectively. The color legend corresponds to the correlation coefficient (r), with darker shades indicating stronger positive or negative correlations.