Genome-Wide Analysis of Staphylococcus aureus Sequence Type 72 Isolates Provides Insights Into Resistance Against Antimicrobial Agents and Virulence Potential

Abstract

Staphylococcus aureus sequence type 72 (ST72) is a major community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) that has rapidly entered the hospital setting in Korea, causing mild superficial skin wounds to severe bloodstream infections. In this study, we sequenced and analyzed the genomes of one methicillin-resistant human isolate and one methicillin-sensitive human isolate of ST72 from Korea, K07-204 and K07-561, respectively. We used a subtractive genomics approach to compare these two isolates to other 27 ST72 isolates to investigate antimicrobial resistance (AMR) and virulence potential. Furthermore, we validated genotypic differences by phenotypic characteristics analysis. Comparative and subtractive genomics analysis revealed that K07-204 contains methicillin (mecA), ampicillin (blaZ), erythromycin (ermC), aminoglycoside (aadD), and tetracycline (tet38, tetracycline efflux pump) resistance genes while K07-561 has ampicillin (blaZ) and tetracycline (tet38) resistance genes. In addition to antibiotics, K07-204 was reported to show resistance to lysostaphin treatment. K07-204 also has additional virulence genes (adsA, aur, hysA, icaABCDR, lip, lukD, sdrC, and sdrE) compared to K07-561, which may explain the differential virulence potential of these human isolates of ST72. Unexpectedly, the virulence potential of K07-561 was higher in an in vivo wax-worm infection model than that of K07-204, putatively due to the presence of a 20-fold higher staphyloxanthin concentration than K07-204. Comprehensive genomic analysis of these two human isolates, with 27 ST72 isolates, and S. aureus USA300 (ST8) suggested that acquisition of both virulence and antibiotics resistance genes by ST72 isolates might have facilitated their adaptation from a community to a hospital setting where the selective pressure imposed by antibiotics selects for more resistant and virulent isolates. Taken together, the results of the current study provide insight into the genotypic and phenotypic features of various ST72 clones across the globe, delivering more options for developing therapeutics and rapid molecular diagnostic tools to detect resistant bacteria.

Keywords: Staphylococcus aureus; antibiotics resistance; sequence type 72; subtractive genomics; virulence factors.

Figure 1

Analysis of isolate-specific distribution of antibiotic resistance and important offensive-virulence genes along with SCCmec and Panton Valentine leucocidin (PVL) in various sequence type 72 (ST72) isolates obtained at different times and locations. (A) Analysis of antibiotics genes in ST72 K07-204 and K07-561 along with 27 other ST72 isolates and ST8 (SAUSA300) revealed variability in their antibiotic resistance. Subtractive genomics allowed identification of isolate-specific virulence genes. The GenBank Accession numbers of each isolates are hyperlinked and ST is indicated in the corresponding rows; (B) Analysis of SCCmec cassette and PVL in various isolates of ST72 and SAUSA300 (ST8). The analysis showed that out of 29 ST72 isolates, only three ST72 isolates are PVL positive while 15 ST72 isolates showed the presence of SCCmec cassette ranging from type IV(2B), IVa(2B), IVc(2B) to V(5C2). ST72 isolates E16SA093 and F17SA003 showed the presence of mecA gene, but no typable SCCmec cassette was identified (orange box).

Figure 2

Experimental validation of the antimicrobial resistance/susceptibility of the human ST72 isolates K07-204 and K07-561. (A) The diagram shows isolate-specific antibiotic resistance gene where the presence of a gene is denoted by a red and black box; (B) Methicillin susceptibility of K07-561 (MSSA) and resistance of K07-204 (MRSA); (C) Variable levels of ampicillin resistance of the two isolates; (D) Erythromycin resistance of K07-204 and susceptibility of K07-561; (E) Variable levels of kanamycin resistance of K07-204 and susceptibility of K07-561; (F) Tetracycline susceptibility of two isolates according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoint table despite the presence of a major facilitator superfamily efflux pump (MFS) tet38; (G) Summary of the resistant/susceptible phenotypes of ST72 isolates (K07-204 and K07-561) and their corresponding MIC₉₀; and (H) Lysostaphin resistance of K07-204 (lys^r) in comparison to the lysostaphin sensitivity of K07-561(lys^s) as reported earlier (Batool et al., 2020a).

Figure 3

Comparative study of the infection potential of K07-204 and K07-561 relative to that of ST8 (S. aureus USA300) under in vitro and in vivo infection conditions, and the possible involvement of major virulence factors. (A,B) K07-204, K07-561, and S. aureus USA300 showing the differential invasion potential in HEK293 cells (A), and phagocytosis and intracellular survival in murine macrophage 264.7 cells (B). (C) In vivo wax-worm infection model showing the comparative pathogenesis of K07-204 and K07-561 with S. aureus USA300. The pathogenesis potential of K07-561 was higher than that of S. aureus USA300. (D,E) Qualitative and quantitative assessment of staphyloxanthin in K07-204 and K07-561 and comparison with S. aureus USA300; K07-204 formed white colonies whereas K07-561 and S. aureus USA300 formed yellow colonies on a TSB agar plate (D); and (E) Quantitation of extracted staphyloxanthin at A_{465 nm}. Both K07-561 and S. aureus USA300 had about 20-fold higher staphyloxanthin levels than K07-204, which could be a possible reason for the equivalent pathogenesis potential of K07-561 and S. aureus USA300 under in vivo conditions.

Figure 4

Phylogenetic analysis of various sequence types, a single nucleotide polymorphism matrix of ST72 isolates, and a phylogram of ST72 showing the phylogenetic affinities of K07-204 and K07-561. (A) Maximum-likelihood phylogeny of 60 isolates based on single nucleotide polymorphisms (SNPs) showing closely related sequence types of ST72. As expected, five representative ST72 isolates comprising K07-204 and K07-561 are grouped together among various sequence types. (B) The SNP matrix of 29 ST72 isolates showing variation among various ST72 isolates; K07-204 and K07-561 differed by 304 SNPs. (C) SNP-based phylogram showing the phylogenetic relationships of K07-204 (blue) and K07-561 (red) and their closely related 27 ST72 isolates wherein K07-204 is closely related with F17SA003 while K07-561 is closely related with two FORC_061 (Korea) and MGYG-HGUT-02337 (Isolation location: NA, submitter EMBL_EBI, Europe, 2019) as both these isolates has no variation with 0 SNP. The “country” and the “year of isolation” of ST72 isolates are shown in bracket wherein NA stands for “not available.”