Plasmid-Mediated mupA Carriage and Clonal Distribution of Staphylococcus aureus: Insights from Whole-Genome Sequencing

Staphylococcus aureus is a major opportunistic pathogen responsible for a spectrum of infections, ranging from superficial skin infections to invasive diseases such as pneumonia, osteomyelitis, bacteremia, and endocarditis [1]. The emergence of methicillin-resistant S. aureus (MRSA) has limited therapeutic options, leading to significant morbidity, mortality, and economic burden globally [2].

Mupirocin, a topical antibiotic that inhibits bacterial isoleucyl-tRNA synthetase (IleRS), is extensively used to eradicate S. aureus carriage in patients and healthcare workers, especially in intensive care units and surgical settings [3]. Its role in decolonization has been crucial in reducing the incidence of hospital-associated MRSA infections. However, the efficacy of mupirocin is being threatened by rising resistance rates [4].

Mupirocin resistance is classified into low-level resistance (LLMR), typically caused by point mutations in the chromosomal ileS gene, and high-level resistance (HLMR), which is generally mediated by acquisition of the mupA or, rarely, mupB gene [5,6]. The mupA gene encodes an alternative IleRS enzyme, rendering mupirocin ineffective at clinically achievable concentrations [7]. Importantly, HLMR is strongly associated with failure of decolonization strategies and has been linked with persistent MRSA outbreaks [8].

Traditional detection methods, including disc diffusion and PCR, are effective in identifying phenotypic resistance and confirming the presence of mupA [9]. However, they provide limited information regarding the genetic context of resistance, such as whether mupA is located on plasmids or integrated into chromosomal elements, the presence of co-resistance genes, or the clonal background of isolates [10].

Next-generation sequencing (NGS) has revolutionized microbial genomics, offering comprehensive insights into resistance determinants, mobile genetic elements, and phylogenetic relationships [11]. Studies from Europe and North America have shown that mupA is often plasmid-borne and associated with insertion sequences such as IS257 and IS431, facilitating horizontal gene transfer [12,13]. Others have demonstrated chromosomal integration of mupA within staphylococcal cassette chromosome (SCC) elements, sometimes co-localized with mecA, highlighting genomic plasticity [14]. Additionally, genomic epidemiology has revealed that mupirocin resistance frequently clusters within epidemic MRSA lineages such as ST239, ST22, and ST5 [15–17].

Despite its clinical importance, genomic characterization of mupirocin resistance remains underexplored in India, where both MRSA prevalence and mupirocin use are high. The present study aimed to investigate the genomic basis of mupirocin resistance in S. aureus isolates from a tertiary-care hospital using NGS, focusing on (i) detection and localization of mupA, (ii) identification of co-resistance genes, and (iii) phylogenetic distribution to assess clonal spread and nosocomial transmission.

Study Isolates

A total of 46 mupirocin-resistant S. aureus isolates, identified phenotypically by disc diffusion, were included.

DNA Extraction and Sequencing

High-quality genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germany). Sequencing libraries were prepared using the Nextera XT kit (Illumina, USA) and sequenced on the Illumina MiSeq platform (2 × 250 bp paired-end reads).

Quality Control and Assembly

Raw reads were assessed using FastQC, and adapters/low-quality reads were trimmed with Trimmomatic. De novo assembly was performed using SPAdes v3.14, yielding draft genomes with average N50 ~65 kb. Annotation was carried out with Prokka v1.14.

Resistance Gene Identification

Resistance determinants were identified using ResFinder 4.1 and the Comprehensive Antibiotic Resistance Database (CARD).

Plasmid Detection

Plasmid-borne genes were identified using PlasmidFinder. Mobile genetic elements such as IS257 and IS431 were identified by BLAST comparison.

Phylogenetic Analysis

Core-genome SNPs were extracted using Snippy pipeline, and phylogenetic trees were generated with RAxML. Sequence types (STs) were assigned using MLST 2.0.

A total of 46 mupirocin-resistant Staphylococcus aureus isolates were subjected to whole-genome sequencing. The findings are presented sequentially, beginning with detection and localization of the mupA gene, followed by identification of co-resistance determinants, phylogenetic distribution of isolates, and ward-specific clustering patterns suggestive of nosocomial transmission.

1. Detection and Localization of mupA

The mupA gene was detected in 32/46 isolates (69.6%). Of these, 22 isolates (73.3%) carried the gene on plasmids, while 10 isolates (26.7%) had chromosomal integrations.

Table 1: Localization of mupA Gene in Mupirocin-Resistant Isolates

Table 1 shows that plasmid-mediated mupA carriage predominated among resistant isolates, reflecting horizontal dissemination.

2. Co-Resistance Genes

Multiple co-resistance genes were detected.

Table 2: Distribution of Co-Resistance Genes Identified by NGS

Table 2 illustrates that beta-lactamase (blaZ) and mecA were common, confirming multidrug resistance among isolates.

3. Phylogenetic Clade Distribution

Table 3: Phylogenetic Distribution of mupA-Positive Isolates

Table 3 shows that most isolates clustered into international epidemic MRSA lineages, suggesting both global clone dissemination and local expansion.

4. Ward-Specific Distribution

Table 4: Distribution of mupA-Positive Isolates Across Hospital Wards

Table 4 demonstrates clustering of mupA-positive isolates in surgery and ICU wards, consistent with nosocomial spread.

This study provides a comprehensive genomic analysis of mupirocin-resistant S. aureus isolates from a tertiary-care hospital, revealing key insights into resistance mechanisms, genetic context, and clonal distribution.

Prevalence and Genetic Basis of Mupirocin Resistance

Among the 46 resistant isolates analyzed, the mupA gene was detected in 69.6%. This finding aligns with studies from Canada, Spain, and the UK, which have reported mupA in 60–80% of high-level resistant isolates [12,18]. The strong association between mupA and high-level mupirocin resistance supports its role as the principal determinant of clinically significant resistance. Notably, 30.4% of resistant isolates lacked mupA, suggesting alternative mechanisms such as ileS mutations, consistent with prior genomic reports [6,19].

Plasmid vs Chromosomal Localization

Our data demonstrated that 73.3% of mupA-positive isolates carried the gene on plasmids, frequently linked with IS257 and IS431, confirming the role of mobile genetic elements in horizontal dissemination. Similar plasmid-mediated mupA carriage has been reported in European hospital outbreaks [12,13]. In contrast, 26.7% of isolates harbored chromosomal integration of mupA within SCC elements, often co-localized with mecA. This dual localization highlights the genomic adaptability of S. aureus, where plasmid-mediated acquisition may evolve into stable chromosomal integration [14,20].

Co-Resistance Genes and Multidrug Resistance

The detection of additional resistance determinants such as blaZ (28.5%), ermC (8.1%), tetK (7.3%), and aac(6′)-Ie-aph(2″)-Ia (6.1%) underscores the multidrug-resistant (MDR) phenotype of these isolates. Previous genomic studies have reported similar co-selection of mupirocin resistance with macrolide, aminoglycoside, and beta-lactam resistance genes [7,21]. The co-occurrence of mupA and mecA in several isolates is particularly concerning, as it indicates strains resistant to both mupirocin-based decolonization and beta-lactam therapy.

Phylogenetic Clustering and Clonal Spread

Core-genome SNP analysis clustered mupA-positive isolates into epidemic lineages ST239, ST22, and ST5, which have been implicated in large-scale MRSA outbreaks worldwide [15–17]. This mirrors findings from genomic surveillance in Europe and Asia, where ST239 (Brazilian/Hungarian clone) and ST22 (EMRSA-15) dominate hospital-acquired MRSA infections [16,22]. Ward-specific clustering in our study suggested nosocomial spread, particularly in surgical and ICU settings, consistent with earlier outbreak reports [23].

Clinical and Epidemiological Implications

The findings of this study have important implications for infection-control strategies. The predominance of plasmid-mediated mupA highlights the risk of rapid horizontal dissemination across strains and wards. Meanwhile, clonal clustering within epidemic MRSA lineages suggests ongoing nosocomial transmission. Together, these mechanisms contribute to the persistence of mupirocin resistance in hospital environments.

Routine surveillance of mupirocin resistance using both phenotypic and molecular approaches should be integrated into hospital antibiograms. In addition, genomic epidemiology should be employed in outbreak investigations to track plasmid-mediated resistance and clonal spread. Restricting indiscriminate mupirocin use and implementing stewardship programs are essential to preserve its effectiveness for decolonization

NGS revealed that mupirocin resistance in S. aureus is largely plasmid-mediated but also associated with chromosomal integration in epidemic MRSA clones. The presence of co-resistance genes and clustering into international lineages emphasize the global relevance of these findings. Genomic surveillance and prudent mupirocin use are essential to curb the spread of resistance.

1.     Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998;339(8):520–532.

2.     Chambers HF, DeLeo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol. 2009;7(9):629–641.

3.     Hetem DJ, Bonten MJ. Clinical relevance of mupirocin resistance in Staphylococcus aureus. J Hosp Infect. 2013;85(4):249–256.

4.     Udo EE, Jacob LE, Mathew B. Genetic analysis of methicillin-resistant Staphylococcus aureus expressing high- and low-level mupirocin resistance. J Med Microbiol. 2001;50(11):909–915.

5.     Seah C, Alexander DC, Louie L, Simor A, Low DE, Longtin J, et al. MupB, a new high-level mupirocin resistance mechanism in Staphylococcus aureus. Antimicrob Agents Chemother. 2012;56(4):1916–1920.

6.     Patel JB, Gorwitz RJ, Jernigan JA. Mupirocin resistance. Clin Infect Dis. 2009;49(6):935–941.

7.     Shore AC, Coleman DC. Mupirocin and mupirocin resistance: a critical appraisal of the literature. Future Microbiol. 2013;8(3):261–274.

8.     McClure JA, Conly JM, Lau V, Elsayed S, Louie T, Hutchins W, et al. Novel mechanism of high-level mupirocin resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 2013;57(11):5041–5050.

9.     Cookson B, et al. Mupirocin-resistant Staphylococcus aureus in the United Kingdom. J Antimicrob Chemother. 1991;27(6):577–586.

10.   Gilbart J, Perry CR, Slocombe B. High-level mupirocin resistance in Staphylococcus aureus: evidence for plasmid transmission in hospitals. Antimicrob Agents Chemother. 1993;37(8):1620–1626.

11.   Simor AE, Stuart TL, Louie L, Watt C, Ofner-Agostini M, Gravel D, et al. Mupirocin-resistant, methicillin-resistant Staphylococcus aureus strains in Canadian hospitals. Antimicrob Agents Chemother. 2007;51(11):3880–3886.

12.   De Lencastre H, Severina EP, Milch H, et al. Dissemination of methicillin- and mupirocin-resistant Staphylococcus aureus clones in Portuguese hospitals. Antimicrob Agents Chemother. 1999;43(2):599–604.

13.   Hodgson JE, Curnock SP, Dyke KG, Morris R, Sylvester DR, Gross MS. Molecular characterization of IS257-mediated integration of mupA plasmids. J Bacteriol. 1994;176(20):5937–5945.

14.   Katayama Y, Ito T, Hiramatsu K. A new class of genetic element, staphylococcal cassette chromosome mec (SCCmec), encodes methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 2000;44(6):1549–1555.

15.   Holden MTG, Lindsay JA, Corton C, et al. Genome sequence of a major epidemic clone of methicillin-resistant Staphylococcus aureus, ST239. Proc Natl Acad Sci U S A. 2010;107(14):6738–6743.

16.   Harris SR, Feil EJ, Holden MT, et al. Evolution of MRSA during hospital transmission and intercontinental spread. Science. 2010;327(5964):469–474.

17.   Peacock SJ, Paterson GK. Mechanisms of methicillin resistance in Staphylococcus aureus. Annu Rev Biochem. 2015;84:577–601.

18.   Eltringham I. Mupirocin resistance and methicillin-resistant Staphylococcus aureus (MRSA). J Hosp Infect. 1997;35(1):1–8.

19.   Hodgson JE, Curnock SP, Dyke KG. Point mutations in ileS associated with low-level mupirocin resistance. J Antimicrob Chemother. 1994;34(5):829–840.

20.   McNeil JC, Hultén KG, Kaplan SL, Mason EO. Plasmid- and chromosomally mediated resistance in MRSA: clinical implications. Antimicrob Agents Chemother. 2011;55(10):4598–4602.

21.   Becker K, Heilmann C, Peters G. Co-resistance in MRSA and impact on therapy. Clin Microbiol Rev. 2014;27(4):870–926.

22.   Grundmann H, et al. Emergence and resurgence of MRSA as a public health threat. Lancet. 2006;368(9538):874–885.

23.   Hetem DJ, et al. Failure of mupirocin-based decolonization of MRSA carriers: clinical implications. Clin Infect Dis. 2016;62(4):491–497.