The Surge of Hypervirulent ST398 MRSA Lineage With Higher Biofilm-Forming Ability Is a Critical Threat to Clinics

Abstract

The global increase of community-associated (CA) infections with methicillin-resistant Staphylococcus aureus (MRSA) is a major healthcare problem. Although sequence type (ST) 398 MRSA was first described as a livestock-associated (LA) lineage, human-adapted MRSA (HO-MRSA) ST398 without livestock contact has subsequently been reported from China in our previous study and other later research. The proportion of ST398 HO-MRSA has also remarkably increased in recent years in China. Based on 3878 S. aureus isolates that were collected in a general hospital between 2008 and 2018, we identified 56 ST398 HO-MRSA isolates. The four early appearing isolates of them have been sequenced by whole-genome sequencing (WGS) in our previous study. Here, by usage of WGS on the later-appearing 52 isolates and analyzing the phylogenetic dynamics of the linage, we found that 50 isolates clustered together with the former 4 isolates, making it a main clade out of MSSA clones and other MRSA clones, although ST398 HO-MRSA evolved with multiple origins. Drug resistance and virulence gene analysis based on the WGS data demonstrated that ST398 HO-MRSA main clade exhibited a similar pattern in both parts. Furthermore, they all carried a conserved variant of prophage 3 to guarantee virulence and a short SCCmec type V element of class D to maintain considerable lower methicillin resistance. Further phenotypical research verified that the epidemic HO-MRSA ST398 displayed enhanced biofilm formation ability when keeping high virulence. The dual advantages of virulence and biofilm formation in the HO-MRSA ST398 subtype promote their fitness in the community and even in the healthcare environment, which poses a serious threat in clinical S. aureus infections. Therefore, further surveillance is required to prevent and control the problematic public health impact of HO-MRSA ST398 in the future.

Keywords: methicillin-resistant staphylococcus aureus; phylogenetic analysis; sequence type 398; virulence; whole-genome sequencing.

FIGURE 1

Prevalence of the total ST398 (A) and the increasingly emerging dominant MRSA ST398 clone (B) from different clinical specimens of patients, 2008–2018.

FIGURE 2

Phylogenetic structure of ST398 S. aureus isolates. (A) Maximum-likelihood phylogenetic tree of ST398. A total of 129 S. aureus ST398 isolates (114 from adult patients and 15 from livestock) were used for phylogenetic reconstruction. Relationships are shown with respect to the genome of the standard LA-MRSA strain (S0385). Branches are colored by geographic origin of isolates. The tree is rooted with ST36 as an outgroup. (B) Unrooted tree of ST398 S. aureus isolates. Branches in this tree could be classified into two main clades, including Clade I and Clade II. Branches are colored by geographic origin of isolates. Black branch: outgroup. Orange branch: reference isolate. Blue branch: human-adapted (HO-MRSA) isolates sequenced in our previous study. Green branch: HO-MSSA isolates in our previous study. Yellow branch: livestock-adapted (LA-MSSA) isolates in our previous study. Red branch: HO-MRSA isolates sequenced in this study.

FIGURE 3

Presence or absence of antibiotic resistance genes and virulence genes among ST398 isolates. The distribution of antibiotic resistance genes and virulence genes is plotted against the core genome phylogeny; the presence of an antibiotic resistance or virulence gene in an isolate is shown in red and blue, respectively, and the absence is shown in gray.

FIGURE 4

Analysis of HO-MRSA ST398 SCCmec. (A) SCCmec comparison. (B) Comparison of oxacillin MIC. p ≤ 0.05 (*).

FIGURE 5

Phenotypic differences between HO-MRSA ST398 in Clade 1 and Clade 2. (A) Hemolysis (erythrocyte lysis) of four representative HO-MRSA ST398 isolates, respectively, in Clade 1 and Clade 2. Hemolytic activities were determined by incubating culture filtrates with human red blood cells. (B,C) Expression of RNAIII/hla was determined by qRT-PCR. (D) Biofilm elaboration measured using polystyrene microtiter plate assay. Statistical analysis was performed using Student’s t-test. The data shown are mean ± SD of three independent experiments. Statistical significances: p > 0.05 (ns), p ≤ 0.05 (*), p < 0.001 (**).