Introduction

Staphylococcus aureus is an important human pathogen and can cause several diseases [1,2], including bacteremia [3]. As such, S. aureus contains a diversity of virulence factors and toxins that favor the development of disease but also allow S. aureus to address challenges presented by the human immune system [4,5].

S. aureus has a high level of resistance acquisition against multiple antibiotic classes, which complicates the treatment of infections associated with this bacterial species [6,7]. Penicillin-resistant epidemic S. aureus strains were followed by the so-called “archaic” methicillin-resistant S. aureus (MRSA) strains first found in the United Kingdom. In the 1980s, novel lineages emerged and have now achieved worldwide distribution [4]. MRSA strains produce an altered penicillin-binding protein, which is encoded by the mecA gene and carried within a mobile genetic element designated staphylococcal cassette chromosome mec (SCCmec) [8].

The Panton-Valentine leukocidin (PVL) is a widely studied toxin, has been associated with neutrophil lysis by the activity of a two component toxin (LukS-PV and LukF-PV) that assemble on neutrophil membranes, leading to lysis. The role for PVL in pathogenesis is unclear [2].

Several methods have been used to describe MRSA lineages, including pulsed-field gel electrophoresis (PFGE) [9], multilocus sequence typing (MLST), spa typing, and SCCmec typing [10]. The information obtained from these methods can be useful in tracing outbreaks. Advances in whole genome sequencing (WGS) technologies allow deeper understanding virulence factors and antimicrobial resistance [11], becoming the method of choice for outbreak analysis of microbial pathogens, including MRSA [11,12].

The use of genomic approaches to develop a genome-wide, gene-by-gene analysis tool includes extended MLST (eMLST) that involves ribosomal MLST (rMLST), core genome MLST (cgMLST), whole-genome MLST (wgMLST), and a pan-genome approach [12,13].

The antimicrobial resistance of S. aureus has significantly varied over time and by geographical location, which highlights the importance and clinical relevance of molecular surveillance for MRSA [14]. Therefore, our study aims to determine the genomic characteristics of MRSA using whole-genome sequencing (WGS) data from medical centers in Mexico and to explore the associations between antimicrobial resistance genes and virulence factors.

Materials and methods

Results

Clinical isolates and susceptibility results

In total, 27 strains were included in this study; most of them were collected from blood (n = 21). All strains were cefoxitin resistant. High MIC values were observed for ciprofloxacin, erythromycin, and clindamycin. In particular, all SCCmec IIa strains showed resistance to levofloxacin and ciprofloxacin, and all but two showed resistance to clindamycin. Complete results for the susceptibility tests are shown in Table 1

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Table 1. Distribution of the SCCmec type, MLST, susceptibility tests among MRSA strains included.

https://doi.org/10.1371/journal.pone.0317284.t001

Clonal complexes, MLST profiling, SCCmec, and spa typing

The most frequent SCCmec detected was IIa (n = 17); all of these were CC5. Moreover, two different STs were detected: ST5 (n = 7) and ST1011 (n = 11), followed by Type IVa (n = 7) and Type V (n = 3; Table 1, Fig 1).

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Fig 1. Whole genome midpoint-rooted SNP phylogenetic tree and antimicrobial resistance-associated genes identified by Pathogenwatch.

The colors to the right of the sample names depict the type of cassette predicted by the SCCmec finder platform.

https://doi.org/10.1371/journal.pone.0317284.g001

Most strains belonged to common and non-overlapping CC; the most important was CC5 (n = 18), which included strains with ST1011 (n = 11), followed by ST5 (n = 7) and SCCmec types IIa (n = 17) and IVa (n = 1; Table 1). They were followed by CC8 with ST8 (n = 3). One strain from CC93 with ST93, and five strains harbored undescribed STs.

Antibiotic results were compared among CC detected. The MIC (Minimum inhibitory concentration) value of ≥8 for levofloxacin was most observed in clonal complex 5 (CC5) compared to other clonal complexes, with a statistically significant p-value of 0.002. No other significant differences were found between the CCs.

Distribution of SCCmec type and predicted antimicrobial susceptibility patterns

In all strains, no resistance was predicted for fusidic acid, vancomycin, linezolid, daptomycin, moxifloxacin, and teicoplanin. Resistance was predicted for most strains of amikacin, tobramycin kanamycin, clindamycin, erythromycin, and ciprofloxacin (see Fig 2).

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Fig 2. Whole genome midpoint-rooted single nucleotide polymorphism phylogenetic tree and antimicrobial susceptibility patterns predicted by Pathogenwatch.

The colors to the right of the sample names depict the type of cassette predicted by the SCCmec finder platform.

https://doi.org/10.1371/journal.pone.0317284.g002

Whole genome SNP phylogenetic tree and virulence genes

The whole genome SNP phylogenetic tree and virulence factors (adhesion, enzymes, immune evasion, secretion systems, and toxins) detected are shown in Fig 3. Multiple genes for adhesion, enzymes, immune evasion, and secretion system were detected, regardless of SCCmec type, including fmbA, fmbB, icaA, icaB, and icaC (adhesion); lop and nuc (enzymes); capsule-related (immune evasion); and esaA, esaB, and esaD (secretion systems).

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Fig 3. Whole genome midpoint-rooted single nucleotide polymorphism phylogenetic tree and virulence factors predicted by Pathogenwatch.

The colors to the right of the sample names depict the type of cassette predicted by SCCmec finder platform.

https://doi.org/10.1371/journal.pone.0317284.g003

By contrast, the detection of toxins showed differences according to SCCmec, with high similarity among strains that harbored the IIa and V SCCmec (both SCCmec harbored several genes, including seg, selm, seln, and selo). The Panton-Valentine leukocidin encoding genes (lukS-PV y lukF-PV) were detected among SCCmec IVa strains.

Comparison with RefSeq genomes

A total of 16,393 genomes were retrieved from the NCBI RefSeq assembly database. After processing with sourmash v4.8.9 [17], 206 genomes (without duplicates) were selected for further analysis. Additional filtering kept only one isolate per BioProject per year, except for samples isolated from sterile sites, where more than one isolate was included. This resulted in a final sample set of eighty isolates (metadata included in S2 Table).

Among these isolates, three different clonal complexes were identified; CC5 was the most frequent [n = 46, STs; 5 (n = 26), 1011 (n = 12), 9034 (n = 3), 105 (n = 2), 225 (n = 1)] untypable (n = 2), followed by CC8 [(n = 31), STs; 8 (n = 29), 9003 (n = 2)] and CC93 (n = 3, ST 93) (Figs 4 and 5).

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Fig 4. Core genome alignment-based maximum likelihood phylogenetic tree of sequenced MRSA isolates and RefSeq similar genomes.

https://doi.org/10.1371/journal.pone.0317284.g004

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Fig 5. Core genome alignment-based maximum likelihood phylogenetic tree of sequenced MRSA isolates and RefSeq similar genomes with branch distances.

The tree scale value represents substitutions per site.

https://doi.org/10.1371/journal.pone.0317284.g005

The mecA gene was detected in seventy-five out of eighty isolates. The five isolates lacking the mecA gene were all part of the CC5 clade and were isolated from sterile sites. Clades were evenly distributed across geographic regions, including America (blue tones), Europe (green tones), and Asia (red tones), as well as across different isolation years. The most recent similar genomes were from Asia (China, 2023, GCF_037911155.1; Taiwan, 2022, GCF_032459935.1) and North America (United States, 2021, GCF_038096265.1). These isolates belonged to the CC8, were isolated from sterile sites, and harbored the mecA gene.

When examining phylogenetic distances (Fig 5) the distance between the CC93 clade and the CC5 and CC8 clades was 0.008 (one mismatch every 125 nucleotides), while the distance between CC5 and CC8 was approximately 0.005 (one mismatch every 200 nucleotides) between them.

Discussion

The molecular epidemiology of S. aureus, including associated antibiotic resistance and virulence factors, change by geographical location. Thus, molecular surveillance for clinically relevant MRSA isolates is key, and must be performed in different populations. In our study, we determined the genomic characteristics of 27 MRSA strains collected in 2022 and 2023 from eight centers in Mexico.

Herein, the most frequent ST found was ST1011, followed by ST5. Both STs had been previously reported in Mexico as a divergent subclade of CC-II A, whose early-branching strains were from the United States and whose most recent common ancestor was estimated to date from the late 1990s [24].

The replacement of CC30 by CC5 was described in Mexico in 2001 [25]. Challagundia et al., previous work on CC5 includes prevalent clones in the Western Hemisphere whose expansion revealed genomic changes toward increased antibiotic resistance and decreased virulence, as shown by the loss of the staphylococcal enterotoxin p (sep) gene from the immune evasion gene cluster of phage ФSa3, which was also absent from the studied MRSA strains [24].

Several studies have reported the molecular characteristics of MRSA isolates in Mexico, including an analysis of 249 S. aureus (MRSA and MSSA) blood isolates collected from pediatric patients at a tertiary care hospital from 2006 to 2019. In this study, 38% of isolates were MRSA, and SCCmec-II was the most frequently detected element (86.3%) [26], as reported in our study (n = 17, 63%). Additionally, the pvl gene was detected in four MRSA SCCmec IV isolates, in which most SCCmec IVa strains harbored the pvl genes.

In another study in Mexico, the molecular characteristics of 51 MRSA isolates collected from a tertiary care hospital between 2017 and 2018 were analyzed. Researchers reported that, most strains harbored the SCCmec type II (76.4%), with ST5-MRSA-II-t895 (New York/Japan clone) and ST1011-MRSA-II-t9364 (New York/Japan-Mexican variant clone). We should note that different lineages of CC5 (85.4%) and CC8 (8.3%) were identified in that study. This study is the first to report the association between the t895 and t9364 spa types and the ST5 and ST1011 lineages [27].

As for our work, we detected both clones with six strains being ST5(CC5)-MRSA-IIa-t895 (New York/Japan clone) and five being the ST1011(CC5)-MRSA-IIa, t9364 (New York/Japan-Mexican variant clone). Other relevant detected strains included ST1011(CC5)-MRSA-IIa, t895 (four strains), and ST8(CC8)-MRSA-IVa, t8 (three strains).

Interestingly, another study from Mexico reported that, of 107 MRSA strains collected from a third-level hospital in Mexico between 2009 and 2010, the most frequently detected SCCmec types were I and II and the ST247 and ST5 [28]. Furthermore, recent studies have documented the emergence of novel genetic lineages of MRSA in Latin America. Particularly, scientists have characterized bloodstream MRSA recovered from nine Latin American countries, including Mexico, with most MRSA isolates being CC5. In fact, CC5 has been shown to be the most MRSA in blood isolates from Latin America (except for Colombia and Ecuador) [29].

The MRSA molecular types reported for Mexico have also been reported for other countries. A molecular epidemiological analysis of MRSA strains isolated from blood in Japan was recently conducted, and this analysis encompassed 88 isolates. The prevalence of SCCmec type II was high in 2014 strains, accounting for a significant portion of the strains studied, however, its presence was significantly reduced in 2018 strains. Conversely, the prevalence of SCCmec type IV strains showed a marked increase, rising from 18.75% in 2014 to 83.87% in 2018. Furthermore, CC5, CC8, and CC1 were detected between 2015 and 2017, with ST1 being dominant [30].

A recent review included reports on the molecular characterization of clinical S. aureus from Malaysia from 2008 to 2020, in which hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA) were included. Among HA-MRSA, the ST22-t032-SCCmec IV MRSA clone was reported to supplant the previous dominating clone, ST239-t037-SCCmec III. Meanwhile, ST30, ST772, ST6, and ST22 were repeatedly detected in CA-MRSA, with none being dominant [31].

An Australian report analyzing 620 MRSA samples collected from three hospitals (two adult and one pediatric) between 2017 and 2021 found that ST93 was the most common strain, corroborating previous Australian studies [32,33]. Our study detected ST5, similar to previous research, and additional STs including ST30, ST72, ST78, ST88, ST93, ST97, and ST239 [34].

In addition, a report examined the molecular characteristics of S. aureus isolated from China in 2021 and 2022, including both MRSA and MSSA. Forty STs were found in 214 clinical S. aureus isolates, with ST59 and ST6697 displaying the most extensive distribution and a higher CC5 frequency [35].

It has been reported that MRSA epidemics have occurred in waves, with fitter and more adaptable lineages replacing previously successful lineages. It is expected that variations may occur across the globe and are associated with different selection pressures that result in epidemiological differences by geographic region. While MRSA possesses a diverse arsenal of toxins, the success of a lineage involves more than just producing toxins but also the acquisition or loss of genetic elements involved in the colonization and adaptation of the strains [5].

The RefSeq database provided information about the global diversity of S. aureus strains, by the inclusion of 53 genomes that combined with our strains give 80 isolates distributed all over the world, including countries from America, Europe, and Asia. In all these strains, CC5 was the most frequent (n = 46), followed by CC8 (n = 31). As expected, the isolates in this study grouped with those from North and South America, predominantly from 2010 to 2015. Interestingly, the most recent similar genomes were from China and the United States, underlying the importance of the high mobility of populations in the present years.

MRSA strains typically harbor the mecA gene, which encodes Penicillin-Binding Protein 2a (PBP2a). This protein is crucial for resistance to methicillin and other extended-spectrum β-lactam antibiotics in MRSA [36]. Previous reports have identified MRSA mecA negative strains that possess a genetic marker, termed mecC, which encodes a trans-peptidase exhibiting a 63% similarity to PBP2a. mecC-containing MRSA strains are often associated with cattle and other animals and can be transferred to human MRSA populations [37]. In our study, the mecA gene was detected in 75 out of the 80 isolates selected from the RefSeq assembly database, and the five isolates lack the mecA gene. All these five strains (CC5 clade) were isolated from sterile sites from humans and are among the genomes retrieved (accession numbers: GCF 030515335.1; GCF 030512085.1; GCF 025505995.1; GCF 022326745.1; GCF 000609765.1).

Our research involved comparing the sequenced isolates to publicly available genomes using multiple bioinformatic tools. The genomes exhibiting the greatest similarity were identified, leading to the selection of a final sample set comprised of 80 isolates representing the most closely related genomes.

In this study, all centers from the Invifar network were invited to participate and only twenty-seven clinical isolates were recovered from sterile fluids in one year; Thus, these strains may not represent the circulating strains in Mexico. All twenty-seven clinical isolates were further analyzed by WGS, with no selection of strains performed. We decided to make this report because there is a lack of information about the clinically relevant circulating clinical isolates analyzed by WGS in Mexico, as noted by the reported MRSA genomes in the https://pubmlst.org/ database, in which only six strains recovered from milk from Mexico were deposited (revised on November 27, 2024). The use of WGS will allow other researchers to continue with analysis of genomes and overall, compare them with other genomes deposited all around the world. Other MRSA clinical isolates were recovered but these strains were collected from sites where normal microbiota may be present; Thus, the clinical relevance of the isolates may be questionable.

This study has several limitations. First, we only included isolates from eight centers in Mexico; thus, differences may be observed in other hospitals or geographic areas of the country. Second, we did not collect any clinical information on patients; thus, no classification of HA-MRSA or CA-MRSA was performed; and third, only 27 strains were included in the study. Hence, it should be noted that the 27 strains represent all strains collected over two years from sterile sites in eight centers.

In conclusion, the most frequent MRSA ST detected was ST1011, followed by ST5. Moreover, the SCCmec elements detected were SCCmec type IIa, followed by type IVa. High MIC values were observed for ciprofloxacin, erythromycin, and clindamycin, particularly within SCCmec IIa. Among the SCCmec IVa strains, most harbored the lukS-PV and lukF-PV genes.

Supporting information