
==== Front
J Infect Dis
J Infect Dis
jid
The Journal of Infectious Diseases
0022-1899
1537-6613
Oxford University Press US

38409272
10.1093/infdis/jiae096
jiae096
Major Article
Bacteria
AcademicSubjects/MED00290
A Comparative Phenotypic and Genomic Analysis of Methicillin-Resistant Staphylococcus aureus ST45 Isolates From Cellulitis and Osteomyelitis in Taiwan
Peng Kuo-Ti Department of Orthopedic Surgery, Chang Gung Memorial Hospital, Chiayi
College of Medicine, Chang Gung University, Taoyuan

Chen Pei-Chun Department of Orthopedic Surgery, Chang Gung Memorial Hospital, Chiayi

Chen Jiun-Liang Department of Orthopedic Surgery, Chang Gung Memorial Hospital, Chiayi

Huang Tsung-Yu Division of Infectious Diseases, Department of Internal Medicine, Chang Gung Memorial Hospital, Chiayi

Peng Yi-Ho National Chia-Yi Girls Senior High School, Chiayi

Liu Ju-Fang School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei

Lee Chiang-Wen Department of Nursing, Division of Basic Medical Sciences, Recurrent Diseases and Health Promotion Research Center, Chang Gung University of Science and Technology, Chiayi
Research Center for Industry of Human Ecology and Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Taoyuan
Department of Rehabilitation, Chang Gung Memorial Hospital, Chiayi

https://orcid.org/0000-0002-6492-0346
Chang Pey-Jium Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan
Department of Nephrology, Chang Gung Memorial Hospital, Chiayi, Taiwan

Correspondence: Pey-Jium Chang, PhD, Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Guishan, Taoyuan 33302, Taiwan, ROC (peyjiumc@mail.cgu.edu.tw).
Potential conflicts of interest . All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

15 9 2024
26 2 2024
26 2 2024
230 3 e568e578
16 10 2023
19 2 2024
24 2 2024
14 3 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.

Abstract

Background

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 45 is a globally disseminated MRSA lineage. Herein, we investigated whether MRSA ST45 isolates from cellulitis and from osteomyelitis display distinctive phenotypic and genomic characteristics.

Methods

A total of 15 MRSA ST45 isolates from cellulitis (CL-MRSA; n = 6) or osteomyelitis (OM-MRSA; n = 9) were collected in a Taiwan hospital. These MRSA ST45 isolates were characterized for their antimicrobial susceptibility, biofilm-forming ability, cellular infectivity in vitro, and pathogenicity in vivo. Four CL-MRSA and 6 OM-MRSA ST45 isolates were selected for whole-genome sequencing (WGS).

Results

Antibiotic resistance tests showed that all OM-MRSA ST45 strains, but not CL-MRSA ST45 strains, were resistant to ciprofloxacin, levofloxacin, gentamicin, and doxycycline. Compared to the CL-MRSA ST45 isolates, the OM-MRSA ST45 isolates had stronger biofilm-forming ability and cellular infectivity and caused more severe disease in mice. WGS analysis revealed that these OM-MRSA ST45 isolates carry multiple common mutations or polymorphisms in genes associated with antibiotic resistance and virulence. Moreover, the transposable elements IS256 and IS257R2 were found only in the OM-MRSA ST45 isolates.

Conclusions

The emergence and spread of the highly pathogenic and multidrug-resistant ST45 MRSAs identified from osteomyelitis may pose a serious threat on public health.

This study examined MRSA ST45 isolates collected from cellulitis (superficial infection) and osteomyelitis (invasive infection) in a Taiwan hospital, and highlighted the phenotypic and genomic differences between isolates causing cellulitis versus osteomyelitis.

MRSA
ST45
cellulitis
osteomyelitis
whole-genome sequencing
Chang Gung Memorial Hospital 10.13039/100012553 CMRPG6J0231-0233 CMRPG6L0441-0443 CORPG6M0051 CORPG6P0011 National Science and Technology Council 10.13039/100020595 109-2314-B-182A-024-MY3 NMRPG6K6011-13
==== Body
pmcMethicillin-resistant Staphylococcus aureus (MRSA) can cause a broad spectrum of diseases, including superficial skin and soft tissue infections and invasive infections such as osteomyelitis, endocarditis, pneumonia, and bacteremia [1, 2]. Currently, it is still a challenge to treat MRSA infections because various MRSA lineages continuously acquire antimicrobial resistance and virulence factors involved in bacterial pathogenesis [3–5].

The MRSA sequence type (ST) 45 is a global MRSA lineage originally found in Berlin in 1993 [6–8] and now widely distributed in North America, Australia, Europe, Asia, and Africa [5, 9, 10]. There are several important MRSA ST45 clones critically associated with nosocomial infections and hospital outbreaks worldwide. In North America, the MRSA ST45 clone carrying staphylococcal cassette chromosome mec (SCCmec) type II (ST45-MRSA-II; also known as USA600) was recognized as a major cause of endocarditis and bloodstream infections [11–13]. Nevertheless, MRSA ST45 strains carrying SCCmec type IV (ST45-MRSA-IV) were commonly found in Germany, the United Kingdom, the Netherlands, Switzerland, and Australia [7, 10, 14]. In Taiwan, MRSA ST45 strain was first observed in 2006 [15]. Later in 2012, a survey study conducted in 14 nursing homes showed that one-fifth of residents and staff members carried MRSA in their nares and that ST45 accounted for 50% of MRSA isolates [16]. Analysis of MRSA isolates from patients visiting the emergency department and healthcare workers in central Taiwan in 2015 also revealed that ST45 strains were the common nasal colonization clone up to 30% of MRSA isolates [17]. Similarly, MRSA strains collected in 2016 from residents and environmental sources in 6 long-term care facilities displayed that about 20% of MRSA isolates were ST45 [18]. More recently, ST45 MRSA was reported as one of the predominant strains associated with skin and soft tissue infection in Taiwan’s custodial facilities in 2017 [19]. These studies suggest that ST45 MRSA is an important clinical pathogen in Taiwan. Of note, most of the MRSA ST45 isolates collected in Taiwan harbored SCCmec type IV or V [16, 17, 20, 21].

In our previous studies, we have shown that MRSA isolates from osteomyelitis (invasive infection) generally exhibited higher antibiotic resistance rates than those from cellulitis (superficial infection) [22]. Due to the clinical impact and high strain diversity of ST45 MRSA, we here aimed to further characterize the phenotypic and genomic features of MRSA ST45 isolates from cellulitis and from osteomyelitis.

METHODS

Patients and Bacterial Isolates

MRSA isolates from cellulitis (n = 104) or from osteomyelitis (n = 110) were obtained at the Chiayi Chang Gung Memorial Hospital in Taiwan between 2017 and 2019. The multilocus sequence typing, SCCmec typing, and spa typing of MRSA isolates were performed as described previously [22]. Duplicate cultures from the same patient were excluded from the study. Clinical and demographic data of patients were collected from the electronic medical records. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (number 201600459B1, 202001374B0, and 202200234B0).

Antibiotic Susceptibility Testing

The minimum inhibitory concentration (MIC) of ciprofloxacin, levofloxacin, fusidic acid, gentamicin, rifampicin, trimethoprim-sulfamethoxazole (TMP-SMX), and vancomycin were determined by Etest as described previously [22]. For measuring the MIC of doxycycline, serial 2-fold dilutions of doxycycline (Sigma-Aldrich) were prepared and then mixed with bacterial suspensions (5 × 105 colony-forming units [CFU]/mL).

Biofilm Formation Assay

Bacterial suspensions (0.5 McFarland) were plated in 96-well plates and incubated at 37°C. After 24 hours of incubation, wells were washed twice with phosphate-buffered saline (PBS), fixed with 99% methanol for 15 minutes, and dried at room temperature. The remaining biofilm was stained with 0.1% crystal violet (Sigma-Aldrich) and then dissolved with 33% glacial acetic acid (Sigma-Aldrich). The optical density of each sample was measured at 570 nm using an enzyme-linked immunosorbent assay reader [23]. The experiments were performed in triplicate and repeated 3 times.

Assessment of Bacterial Internalization by Confocal Microscopy

Mouse pre-osteoblast cell line MC3T3-E1 were cultured in medium without or with antibiotics containing 50 units/mL penicillin and 50 µg/mL streptomycin. Bacterial isolates were added at a multiplicity of infection of 100 and incubated at 37°C. At the indicated time points after infection, cells were washed twice with PBS and then fixed in 4% paraformaldehyde solution for 15 minutes. The fixed cells were permeabilized with 0.1% Triton X-100 for 10 minutes, and then incubated with anti–S aureus antibody (Abcam) overnight at 4°C. Slides were then stained with antimouse Alexa 488 (Jackson ImmunoResearch), DAPI, and phalloidin Alexa 633 (Invitrogen), and analyzed using Leica SP5 II confocal microscope. The experiments were performed in duplicate and repeated 3 times.

Animal Experiments

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Chang Gung Memorial Hospital (number 2022021601). For skin infection, 1 × 107 CFU of S aureus were subcutaneously injected into the back skin of C57BL/6J mice. The bacterial growth in mice was analyzed by fluorescence molecular tomography (FMT) using FMT4000 in vivo imaging system (PerkinElmer). Prior to FMT imaging, 100 μL of IVISense Bacterial Detection Probe 750 (PerkinElmer) was intravenously injected into mice for 2 hours. Lesion size of skin was measured at 48 hours.

For bone infection, C57BL/6J mice were infected with 1 × 108 CFU of S aureus as described previously [24]. Bone X-ray images were used to assess the severity of bone destruction. Bone destruction was scored 0 to 4 according to the following criteria: 0, intact bone; 1, mild bone destruction; 2, moderate bone erosion; 3, permeative bone loss; 4, pathologic fracture.

Whole-Genome Sequencing and Analysis

DNA libraries of MRSA isolates were prepared and sequenced by both the Illumina MiSeq system (with 2 × 301 bp paired-end reads) and Nanopore GridION system (using FLO-MIN106 flow cells). The quality of raw sequence data was checked with CLC Genomics Workbench v10 and NanoPlot v1.28.1, respectively. After read trimming, the whole-genome sequences of individual MRSA isolates were assembled with SPAdes v313.0. All sequencing data of MRSA ST45 strains are available at National Center for Biotechnology Information (accession ID: PRJNA944816). For analyzing genetic variations among MRSA ST45 strains, sequence comparisons were performed against the strain sta-747 using default settings of BLASTN.

Phylogenetic Analysis

A phylogenetic tree was constructed using MRSA ST45 isolates in the study (n = 10) and previously reported MRSA ST45 isolates (n = 91). The MRSA ST45 strain CA-347 (accession number CP006044) served as the reference strain. The phylogenetic tree was constructed by the neighbor-joining method using the CLC Genomics Workbench v10 software. The Interactive Tree Of Life (http://itol.embl.de) was used to manipulate and annotate the phylogenetic tree.

Statistical Analysis

Data are expressed as mean ± standard deviation (SD) and the Student t test was used to evaluate differences between samples. A P value <.05 was considered statistically significant.

RESULTS

Clinical Characteristics of MRSA ST45 Isolates From Cellulitis and Osteomyelitis

Among 214 MRSA isolates from cellulitis or osteomyelitis, 15 MRSA isolates were characterized as the ST45 lineage, including 6 isolates from cellulitis (CL-MRSA) and 9 isolates from osteomyelitis (OM-MRSA). The basic demographics of patients and the molecular typing of these ST45 isolates are shown in Table 1. The CL-MRSA ST45 isolates were mainly obtained from male patients, whereas the OM-MRSA ST45 isolates were mainly from female patents. There were no statistically significant differences in patient age and body mass index between the 2 types of infection (Table 1). Both CL-MRSA and OM-MRSA ST45 infections were predominantly observed in limbs of patients, and diabetes was the most common comorbidity. Molecular typing analysis revealed that all CL-MRSA ST45 isolates carried SCCmec type IV; however, all OM-MRSA ST45 isolates carried SCCmec type V. The spa typing analysis showed that the majority of the CL-MRSA ST45 isolates were t026 (67% [n = 4/6]), whereas all OM-MRSA ST45 isolates were t1081 (100% [n = 9/9]).

Table 1. Patient Demographics and the Molecular Typing of Methicillin-Resistant Staphylococcus aureus Sequence Type 45 Isolates

Strain	Collection Year	Sex	Age, y	BMI, kg/m2	Infection Site	Specimena	Comorbidity	SCCmec	spa Type	WGS	
Cellulitis	
 sta-747	2018	M	50	39.2	Nose	Pus swab	Gout	IV	t026	Yes	
 sta-700	2018	M	66	23.6	Thumb	Pus swab		IV	t026	Yes	
 sta-825	2018	F	9	19.2	Gingival	Pus swab		IV	t026	Yes	
 sta-677	2018	F	88	24	Leg	Pus swab	DM, CKD	IV	t1081	Yes	
 sta-1604	2019	M	47	32.8	Toe	Wound swab	Gout, CKD	IV	t026	No	
 sta-1697	2019	M	89	24.6	Ankle	Wound swab	DM, CKD, CHC	IV	t1081	No	
Osteomyelitis	
 sta-P17	2017	F	66	19	Metatarsal	Wound swab	DM	V	t1081	Yes	
 sta-165	2017	M	79	19.6	Phalange	Wound swab	Gout	V	t1081	Yes	
 sta-216	2017	F	32	29.2	Tibia	Pus swab	…	V	t1081	Yes	
 sta-252	2017	M	62	23.9	Sternum	Wound swab	DM	V	t1081	Yes	
 sta-301	2017	M	62	17.8	Sternum	Wound swab	DM	V	t1081	Yes	
 sta-726	2018	F	89	26	Femur	Pus swab	…	V	t1081	Yes	
 sta-1628	2019	F	77	22.8	Femur	Wound swab	DM	V	t1081	No	
 sta-1753	2019	F	68	24.4	Tibia	Wound swab	…	V	t1081	No	
 sta-1850	2019	F	68	33.9	Tibia	Pus swab	…	V	t1081	No	
Abbreviations: BMI, body mass index; CHC, chronic hepatitis C; CKD, chronic kidney disease; DM, diabetes mellitus; F, female; M, male; MRSA, methicillin-resistant Staphylococcus aureus; WGS, whole-genome sequencing.

aSample collection for bacterial culture.

Antibiotic Resistance Profiles of CL-MRSA and OM-MRSA ST45 Isolates

Antibiotic resistance tests revealed that all OM-MRSA ST45 strains, but not CL-MRSA ST45 strains, displayed resistance to ciprofloxacin (MIC ≥12 μg/mL), levofloxacin (MIC ≥2 μg/mL), gentamicin (MIC ≥8 μg/mL), and doxycycline (MIC ≥2 μg/mL) (Table 2). In particular, 2 OM-MRSA ST45 strains including sta-252 and sta-301 conferred resistance to fusidic acid (MIC ≥4 μg/mL) (Table 2). None of the MRSA ST45 isolates were resistant to rifampicin (MIC ≥4 μg/mL), TMP-SMX (MIC ≥4 μg/mL), or vancomycin (MIC ≥2 μg/mL).

Table 2. Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus aureus Sequence Type 45 Isolates From Cellulitis and Osteomyelitis

Strain	MIC, μg/mL	
Ciprofloxacin	Levofloxacin	Fusidic Acid	Gentamicin	Rifampicin	TMP-SMX	Vancomycin	Doxycycline	
Cellulitis									
 sta-747	0.19	0.064	0.047	0.19	0.008	0.047	0.5	0.125	
 sta-700	0.19	0.064	0.047	0.38	0.006	0.047	0.38	0.125	
 sta-825	0.19	0.094	0.032	0.19	0.004	0.047	0.5	0.125	
 sta-677	0.19	0.19	0.047	0.25	0.008	0.047	0.75	0.125	
 sta-1604	0.25	0.19	0.032	0.38	0.012	0.064	1	0.125	
 sta-1697	0.19	0.125	0.032	0.25	0.008	0.064	0.75	0.125	
Osteomyelitis									
 sta-P17	16	3	0.064	16	0.006	0.064	1	2	
 sta-165	16	3	0.032	12	0.008	0.047	0.5	2	
 sta-216	16	2	0.032	16	0.008	0.047	0.75	2	
 sta-252	16	3	4	8	0.004	0.047	0.75	2	
 sta-301	16	3	4	8	0.004	0.094	1	4	
 sta-726	12	2	0.064	12	0.006	0.047	0.75	2	
 sta-1628	12	4	0.19	0.38	0.008	0.064	0.5	2	
 sta-1753	24	6	0.064	6	0.008	0.047	0.5	2	
 sta-1850	16	8	0.064	4	0.04	0.047	0.5	2	
Abbreviations: MIC, minimum inhibitory concentration; TMP-SMX, trimethoprim-sulfamethoxazole.

Biofilm-Forming Ability and Cell Infectivity of MRSA ST45 Isolates

The biofilm-forming assay found that OM-MRSA ST45 isolates had a stronger ability to produce biofilms than CL-MRSA ST45 isolates (Table 3, 1.08 ± 0.53 vs 0.55 ± 0.11; Supplementary Figure 1). To compare the infectivity of these MRSA isolates, the bacterial internalization into mouse MC3T3-E1 cells was measured by confocal microscopy. In the initial evaluation, we found that different MRSA ST45 strains displayed variable internalization rates in MC3T3-E1 cells within 30–60 minutes of incubation in the absence of antibiotics (Supplementary Figure 2A). We therefore compared the bacterial internalization of MRSA ST45 isolates at 45 minutes postinfection. Our results revealed that OM-MRSA ST45 strains exhibited higher percentages of infected cells and higher numbers of internalized bacteria per cell than CL-MRSA ST45 strains (Table 3 and Supplementary Figure 2B). When penicillin and streptomycin were added to cell culture media, we also found that OM-MRSA ST45 strains had higher infectivity than CL-MRSA ST45 strains (Table 3 and Supplementary Figure 3).

Table 3. Biofilm-Forming Ability and Cell Infectivity of Methicillin-Resistant Staphylococcus aureus Sequence Type 45 Isolates From Cellulitis and Osteomyelitis

Strain	Biofilma	Infectivity (Without Antibiotics)b	Infectivity (With Antibiotics)c	
Infected Cells, %	No. of Bacteria/Cell	Infected Cells, %	No. of Bacteria/Cell	
Cellulitis						
 sta-747	0.64 ± 0.08	30.5 ± 6.9	1.56 ± 0.33	20.8 ± 14.6	0.61 ± 0.25	
 sta-700	0.56 ± 0.01	20.8 ± 4.1	1.49 ± 0.26	14.9 ± 3.0	1.29 ± 0.60	
 sta-825	0.51 ± 0.03	13.2 ± 5.5	0.92 ± 0.15	23.7 ± 2.2	0.92 ± 0.10	
 sta-677	0.67 ± 0.02	25.8 ± 4.4	1.00 ± 0.15	31.7 ± 1.7	1.85 ± 0.65	
 sta-1604	0.37 ± 0.05	27.9 ± 7.4	1.91 ± 0.28	22.7 ± 2.1	0.49 ± 0.14	
 sta-1697	0.64 ± 0.03	27.7 ± 9.1	1.80 ± 0.73	22.6 ± 6.6	0.69 ± 0.34	
 Mean ± SD	0.55 ± 0.11	24.3 ± 8.5	1.45 ± 0.37	22.7 ± 5.0	0.98 ± 0.47	
Osteomyelitis						
 sta-P17	1.09 ± 0.38	38.3 ± 8.3	4.74 ± 1.38	30.0 ± 4.7	1.01 ± 0.19	
 sta-165	0.88 ± 0.26	58.0 ± 17.4	3.67 ± 1.34	58.8 ± 12.6	2.27 ± 0.58	
 sta-216	1.50 ± 0.26	91.7 ± 8.3	6.28 ± 0.72	41.1 ± 16.1	1.32 ± 0.32	
 sta-252	0.77 ± 0.03	37.0 ± 14.1	5.45 ± 1.20	56.9 ± 3.1	3.15 ± 0.15	
 sta-301	0.81 ± 0.09	38.4 ± 5.2	4.18 ± 1.18	52.3 ± 2.3	2.29 ± 0.11	
 sta-726	2.50 ± 0.02	51.8 ± 10.7	5.41 ± 1.62	52.5 ± 2.5	4.24 ± 0.70	
 sta-1628	0.98 ± 0.11	50.3 ± 4.1	4.81 ± 1.08	32.2 ± 3.0	2.32 ± 0.81	
 sta-1753	0.70 ± 0.23	68.6 ± 11.4	5.07 ± 0.07	42.6 ± 6.9	2.07 ± 0.40	
 sta-1850	0.73 ± 0.11	50.4 ± 6.3	4.96 ± 0.67	29.1 ± 11.7	1.18 ± 0.96	
 Mean ± SD	1.08 ± 0.53	53.8 ± 16.5	4.95 ± 0.71	43.8 ± 11.1	2.21 ± 0.97	
P value	<.001	.002	<.001	.001	.01	
Abbreviations: SD, standard deviation.

aOptical density (OD600) analysis of crystal violet staining of biofilm (n = 3).

bBacterial internalization evaluated at 45 minutes postinfection in the absence of antibiotics (n = 3).

cBacterial internalization evaluated at 180 minutes postinfection in the presence of antibiotics (n = 3).

Analysis of the Pathogenicity of MRSA ST45 Isolates in Animal Models

To determine the pathogenicity of CL-MRSA and OM-MRSA ST45 isolates in vivo, mouse models of skin infection and bone infection were used. In the skin infection model, FMT analysis revealed that the OM-MRSA group displayed higher bacterial fluorescence intensities than the CL-MRSA group at 24 hours postinfection (Table 4 and Supplementary Figure 4A). However, at 48 hours postinfection, the fluorescent intensities of both the CL-MRSA and OM-MRSA groups were comparable. Notably, we noticed that infection by the OM-MRSA group was significantly associated with larger sizes of skin lesions in mice as compared to infection by the CL-MRSA group (Table 4 and Supplementary Figure 4B). In the model of bone infection, FMT images showed that the overall bacterial growth of OM-MRSA ST45 strains in bone was better than that of CL-MRSA ST45 strains at day 2 and day 6 after infection (Table 4 and Supplementary Figure 5A). Radiographic evaluation also revealed that OM-MRSA ST45 strains caused more severe disease than CL-MRSA ST45 strains (Table 4 and Supplementary Figure 5B).

Table 4. Pathogenicity and Virulence of Methicillin-Resistant Staphylococcus aureus Sequence Type 45 Isolates From Cellulitis and Osteomyelitis

Strain	Pathogenicitya (Skin Infection)	Pathogenicitya (Bone Infection)	
FMT (24 h)	FMT (48 h)	Lesion Sizeb, cm2	FMT (2 d)	FMT (6 d)	Scorec	
Cellulitis							
 sta-747	573 ± 491	1184 ± 63	0.15 ± 0.15	398 ± 1	1426 ± 981	2.5 ± 0.5	
 sta-700	378 ± 158	1270 ± 20	0.00 ± 0.00	436 ± 286	1032 ± 255	1.7 ± 0.5	
 sta-825	769 ± 76	1620 ± 120	0.00 ± 0.00	846 ± 114	1079 ± 635	2 ± 0.8	
 sta-677	441 ± 187	1145 ± 45	0.00 ± 0.00	756 ± 216	884 ± 73	1.5 ± 0.5	
 sta-1604	671 ± 31	1458 ± 8	0.00 ± 0.00	ND	ND	ND	
 sta-1697	693 ± 137	846 ± 46	0.15 ± 0.15	ND	ND	ND	
 Mean ± SD	587 ± 139	1304 ± 252	0.05 ± 0.11	609 ± 440	1102 ± 289	1.9 ± 0.7	
Osteomyelitis							
 sta-P17	811 ± 16	1879 ± 23	1.00 ± 0.00	933 ± 17	1682 ± 551	3 ± 1	
 sta-165	625 ± 21	1267 ± 17	1.00 ± 0.00	710 ± 7	1290 ± 243	3 ± 0.8	
 sta-216	1253 ± 75	1667 ± 17	1.00 ± 0.00	1335 ± 28	2060 ± 53	3.5 ± 0.5	
 sta-252	1105 ± 7	1471 ± 56	0.85 ± 0.05	1829 ± 267	2256 ± 956	3.5 ± 0.5	
 sta-301	1439 ± 15	1383 ± 33	1.10 ± 0.10	1292 ± 531	1485 ± 640	3.3 ± 0.5	
 sta-726	1029 ± 371	1205 ± 5	1.40 ± 0.10	1961 ± 239	2077 ± 20	3.5 ± 0.5	
 sta-1628	1160 ± 844	1569 ± 19	0.25 ± 0.25	ND	ND	ND	
 sta-1753	1054 ± 6	1120 ± 20	0.50 ± 0.30	ND	ND	ND	
 sta-1850	964 ± 386	1440 ± 315	0.35 ± 0.05	ND	ND	ND	
 Mean ± SD	1049 ± 238	1450 ± 247	0.83 ± 0.39	1343 ± 488	1808 ± 632	3.3 ± 0.7	
P value	<.001	.08	<.001	.001	.013	.001	
Abbreviations: FMT, fluorescence molecular tomography; ND, not determined; SD, standard deviation.

aThe fluorescence intensity (pmol) of labeled bacteria in mice quantified by FMT at the indicated time points after infection (n = 3).

bLesion size in mice evaluated at 48 hours postinfection (n = 3).

cBone destruction scores (scale 0–4) evaluated in mice after 6 days of infection (n = 3).

Whole-Genome Sequence of MRSA ST45 Isolates

For whole-genome sequencing (WGS), all MRSA ST45 isolates collected in 2017–2018 were selected (Table 1). These included 4 CL-MRSA ST45 isolates (sta-747, sta-700, sta-825, and sta-677) and 6 OM-MRSA ST45 isolates (sta-P17, sta-165, sta-216, sta-252, sta-301, and sta-726) (Table 1). The genome sizes of these MRSA ST45 isolates ranged from 2.8 to 2.9 Mb (Supplementary Table 1). The G + C contents of genomes in these MRSA ST45 isolates were about 32.8%, and the numbers of annotated open reading frames in these ST45 strains ranged from 2618 to 2734 (Supplementary Table 2).

Phylogenetic Analysis of MRSA ST45 Isolates

In addition to 10 MRSA ST45 isolates in the study, 91 MRSA ST45 isolates mainly from Singapore, Australia, Germany, the United States, Denmark, the United Kingdom, and other areas in Taiwan were included in the comparison of genome sequences (Supplementary Table 3). Based on the phylogenetic analysis, these MRSA ST45 population could be divided into 2 major groups, I and II (Figure 1 and Supplementary Figure 6). Group I contained strains mainly from Taiwan, Singapore, and Australia, and all OM-MRSA ST45 strains were included in this group (Figure 1, right side). It is noteworthy that the CL-MRSA strain sta-677 also belonged to group I. Strains in group II were mainly isolated from Europe, the United States, and Taiwan (Figure 1, left side). Three CL-MRSA ST45 strains, including sta747, sta-825, and sta-700, belonged to this group (Figure 1, left side).

Figure 1. Rooted phylogenetic tree of 101 methicillin-resistant Staphylococcus aureus (MRSA) ST45 isolates based on single-nucleotide polymorphisms in the core genomes. In addition to 10 MRSA ST45 isolates including 4 from cellulitis (CL-MRSA) and 6 from osteomyelitis (OM-MRSA) presented in this study, 91 MRSA ST45 isolates mainly from Singapore, Australia, Germany, USA, Denmark, UK, and other areas in Taiwan were included in this analysis. The CA-347 strain isolated from USA was used as a reference. The scale bar indicates the number of nucleotide substitutions per site.

Analysis of Genomic Variations Among MRSA ST45 Isolates

To investigate the genomic variations among MRSA ST45 isolates, specific antimicrobial resistance (AMR)–related genes, virulence-related genes, transposable elements, prophages, and plasmids within bacteria were analyzed. In the comparison, the strain sta-747 was used as a reference.

AMR-Related Genes

For the resistance to fluoroquinolone (ciprofloxacin and levofloxacin), an S80F substitution in GrlA (DNA topoisomerase IV subunit A) and an S84L substitution in GyrA (DNA gyrase subunit A) [25–27] were found only in OM-MRSA strains (Figure 2 and Supplementary Table 4). Additionally, a D641E substitution in GrlB (DNA topoisomerase IV subunit B) was also specifically detected in OM-MRSA strains (Figure 2 and Supplementary Table 4). For the resistance to fusidic acid, 2 OM-MRSA strains including sta-252 and sta-301 acquired the resistance genes fusB and fusC (Figure 2 and Supplementary Table 4). For the resistance to gentamicin, we could not find mutations in genes encoding 30S ribosome subunits or acquisition of antibiotic resistance genes such as aph(3′), acc(6′), and ant(4′) in OM-MRSA strains. However, specific amino acid substitutions in TrmD (tRNA methyl transferase) and AtpB (ATP synthase subunit B) [28, 29] were detected only in OM-MRSA strains (Figure 2 and Supplementary Table 4). The resistance to doxycycline for OM-MRSA strains could be associated with the acquisition of tetK gene in their genome (Figure 2 and Supplementary Table 4). As expected, all MRSA ST45 strains contained mecA, but not the vanA resistance gene. Ten efflux pump systems including norA, norB, norC, lmrS, mdeA, sdrM, mepA, sav1866, abcA, and sepA [30] were also analyzed. Among them, specific substitution (G470V) in MdeA was detected only in OM-MRSA strains (Figure 2 and Supplementary Table 4).

Figure 2. Genetic variations of antimicrobial resistance–related genes and virulence-related genes in the methicillin-resistant Staphylococcus aureus ST45 isolates from cellulitis and osteomyelitis.

Virulence-Related Genes

Different virulence-related genes involved in (i) bacterial adherence, invasion, and persistence (biofilm formation), (ii) toxin biosynthesis, (iii) immune evasion, and (iv) the regulatory systems influencing virulence gene expression were analyzed. Sequence comparison displayed that the CL-MRSA strain sta-677 and all 6 OM-MRSA strains often shared identical mutations or substitutions in virulence-related genes (Figure 2 and Supplementary Table 5). Only a small number of mutations or substitutions in virulence-related genes were specifically found in OM-MRSA strains, but not in CL-MRSA strains. For example, a H231Q substitution in Emp (extracellular matrix protein) and a V88F substitution in HlgA (γ-hemolysin A) were only detected in OM-MRSA strains (Figure 2 and Supplementary Table 5). Particularly, we noticed that several staphylococcal enterotoxin genes such as seg, sei, sel, sem, sen, and seo were predominantly present in CL-MRSA strains (Figure 2 and Supplementary Table 5).

Transposable Elements, Prophages, and Plasmids

Multiple types of transposable elements were found in the genomes of MRSA ST45 isolates (Supplementary Table 6). Among them, IS256 and IS257R2 were found only in OM-MRSA strains, whereas IS1272 was found only in CL-MRSA strains. There were a total of 19–24 copies of IS256 and 2 copies of IS257R2 in the genomes of OM-MRSA strains (Figure 3 and Supplementary Table 6). Intriguingly, IS1272 insertion was found at the same position within all 4 CL-MRSA genomes, whereas 9 insertions of IS256 and 2 insertions of IS257R2 were found at the same positions within all 6 OM-MRSA genomes (Figure 3 and Supplementary Table 7). These 9 insertions of IS256 were located at 1080k, 1130k, 1174k, 1398k, 1457k, 1463k, 1555k, 1692k, and 1895k within the bacterial genome, and the 2 insertions of IS257R2 were located at 818k and 823k (Figure 3). Multiplex polymerase chain reaction analysis (primer sets in Supplementary Table 8) showed that all collected OM-MRSA ST45 strains (n = 9) contained IS256 insertions at these 9 sites in their genomes (Supplementary Figure 7). Herein, we could not find a significant relationship between prophage profiles and phenotypic features of these MRSA ST45 isolates (Supplementary Table 6). Except for sta-747, all sequenced MRSA ST45 strains contained a plasmid. The plasmid in all 6 OM-MRSAs was 3 kb long, potentially encoding 3 proteins. However, the plasmid in CL-MRSAs was 8 kb, potentially encoding 7–8 proteins (Supplementary Tables 2 and 6).

Figure 3. Schematic diagram of IS1272 insertion in the genome of methicillin-resistant Staphylococcus aureus ST45 isolates from cellulitis (CL-MRSA) and the insertion of IS256 and IS257R2 in the genome of methicillin-resistant S aureus ST45 from osteomyelitis (OM-MRSA) isolates. The insertion positions of IS1272, IS256, and IS257R2 in bacterial genomes are shown as lollipops. The lollipops with a red arrow at the top highlight the insertions of transposable elements occurring at same positions in the genome of CL-MRSA or OM-MRSA ST45 isolates.

DISCUSSION

In Taiwan, the MRSA ST45 lineage is becoming a dominant colonization clone in healthcare workers, patients, and environments of nursing homes, long-term care facilities, and hospitals, accounting for 20%–50% of MRSA isolates [16–18]. The high colonization rates of MRSA ST45 strains may pose a high risk of clinical infections. Due to the high diversity of MRSA ST45 strains, certain MRSA ST45 strains may cause serious life-threatening diseases. In this study, although only 7% (n = 15/214) of MRSA isolates from cellulitis or osteomyelitis were identified as the ST45 lineage, the occasional spread of these clinically pathogenic isolates may result in a serious threat to public health.

Generally, deep tissue infections (such as osteomyelitis) by MRSAs are more severe and more difficult to treat than superficial tissue infections (such as cellulitis) by MRSAs. Herein, we show that there are noticeable differences in the genetic and phenotypic features between CL-MRSA and OM-MRSA ST45 isolates. Molecular typing revealed that CL-MRSA ST45 strains mainly harbored SCCmec IV and t026; however, all 9 OM-MRSA ST45 strains carried SCCmec V and t1081 (Table 1). Compared to the CL-MRSA ST45 strains, the OM-MRSA ST45 strains showed high-level resistance to ciprofloxacin, levofloxacin, gentamicin, and doxycycline (Table 2). Additionally, the OM-MRSA ST45 strains exhibited stronger biofilm-forming ability and cellular infectivity in vitro, and caused more severe disease in mice than the CL-MRSA ST45 strains (Tables 3 and 4). All of these findings suggest that these OM-MRSA ST45 strains are highly pathogenic strains.

To compare genetic variations, high-quality genome sequences of 4 CL-MRSAs and 6 OM-MRSAs were obtained by using the combination of both Illumina MiSeq sequencing and Oxford Nanopore sequencing systems. According to the phylogenetic analysis, all sequenced OM-MRSAs and a CL-MRSA strain sta-677 could be categorized into the same group, whereas the other 3 CL-MRSAs (including sta-747, sta-825, and sta-700) were categorized into another group (Figure 1). Due to the increased use of different kinds of antibiotics to treat bacterial infections, increasing resistance to these antimicrobials takes place in MRSAs. Fluoroquinolones, such as ciprofloxacin and levofloxacin, are the major class of antibiotics that directly inhibit DNA synthesis in bacteria [25]. The primary target of fluoroquinolones is topoisomerase IV (encoded by the grlA and grlB genes), whereas the secondary target is DNA gyrase (encoded by the gyrA and gyrB genes) [26, 27]. In the present study, S80F substitution in GrlA, D641E substitution in GrlB, and S84L substitution in GyrA were detected only in OM-MRSAs (Figure 2). Fusidic acid is usually combined with other antibiotics such as vancomycin or teicoplanin to treat MRSA infections. We found that the fusidic acid–resistant strains sta-252 and sta-301 carried both fusB and fusc (Figure 2) [31, 32]. We additionally proposed that specific amino acid substitutions in TrmD and AtpB could contribute to gentamicin resistance in OM-MRSA ST45 isolates [28, 29], whereas acquisition of tetK gene in OM-MRSA ST45 isolates could cause doxycycline resistance [33]. Although several variants of efflux pump genes have been detected in MRSA ST45 isolates, the major role of these gene variants in antimicrobial resistance needs to be further investigated.

In addition to antimicrobial resistance genes, multiple common mutations (or polymorphisms) occurred in virulence-related genes of OM-MRSA ST45 strains. Despite such, the association of these genetic variants with the pathogenicity of MRSA ST45 isolates still remains obscure. Notably, analysis of transposable elements in bacterial genomes showed that multiple copies of IS256 (19–24 copies) and IS257R2 (2 copies) were found only in OM-MRSA ST45 isolates. Both IS256 and IS257R2 are highly active insertion sequence elements previously identified in the genomes of enterococci and staphylococci, and may affect the expression of virulence and drug resistance genes [34–38]. Importantly, 9 copies of IS256 and 2 copies of IS257R2 were inserted at the same positions in the genomes of all 6 OM-MRSA strains (Figure 3). Although the precise role of IS256 and IS257R2 insertions in OM-MRSA ST45 strains remains unclear, we think that certain insertions of IS256 or IS257R2 may be partly associated with the bacterial antimicrobial resistance or pathogenicity. Clinically, these transposable elements may serve as a source of genetic markers to differentiate OM-MRSA from CL-MRSA ST45 strains.

Overall, this study shows significant differences in the phenotypic and genomic characteristics between CL-MRSA and OM-MRSA ST45 isolates. Understanding the strain diversity of MRSA ST45 isolates may be important for improving empirical antibiotic therapy in patients. However, this study has several limitations. First, only a small number of CL-MRSA and OM-MRSA ST45 isolates were included in the phenotypic and genomic comparison. Second, mutations or polymorphisms in noncoding regions (such as promoters) of virulence-related genes have not been analyzed. Third, the temporal expression profiles of MRSA virulence-related genes during infection remains largely unclear. Therefore, more in-depth investigations with larger sample sizes of MRSA ST45 isolates are needed for future research.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Supplementary Material

jiae096_Supplementary_Data

Notes

Acknowledgments. We thank the Laboratory Animal Center, Department of Medical Research, Chang Gung Memorial Hospital at Chiayi for animal experiments, and Precious Instrumentation Core Laboratory, Department of Medical Research, Chang Gung Memorial Hospital at Chiayi for technical assistance of confocal microscope.

Financial support . This work was supported by Chang Gung Memorial Hospital, Chiayi, Taiwan (grant numbers CMRPG6J0231-0233, CMRPG6L0441-0443, CORPG6M0051, and CORPG6P0011); and the Ministry of Science and Technology, Taiwan (grant numbers 109-2314-B-182A-024-MY3, 112-2314-B-182A-099 and NMRPG6K6011-13). Funding to pay the Open Access publication charges for this article was provided by Chang Gung Memorial Hospital.
==== Refs
References

1 Hiramatsu K , CuiL, KurodaM, ItoT. The emergence and evolution of methicillin-resistant Staphylococcus aureus. Trends Microbiol 2001; 9 :486–93.11597450
2 Lakhundi S , ZhangK. Methicillin-resistant Staphylococcus aureus: molecular characterization, evolution, and epidemiology. Clin Microbiol Rev 2018; 31 :e00020-18.30209034
3 Grundmann H , Aires-de-SousaM, BoyceJ, TiemersmaE. Emergence and resurgence of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet 2006; 368 :874–85.16950365
4 Copin R , SauseWE, FulmerY, et al Sequential evolution of virulence and resistance during clonal spread of community-acquired methicillin-resistant Staphylococcus aureus. Proc Natl Acad Sci U S A 2019; 116 :1745–54.30635416
5 Mohamad Farook NA , ArgimonS, Abdul SamatNM, et al Diversity and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) genotypes in Southeast Asia. Trop Med Infect Dis 2022; 7 :438.36548693
6 Witte W . Antibiotic resistance in gram-positive bacteria: epidemiological aspects. J Antimicrob Chemother 1999; 44 (Suppl A ):1–9.
7 Witte W , GuidoW, CunyC. Subtyping of MRSA isolates belonging to a widely disseminated clonal group by polymorphism of the dru sequences in mec-associated DNA. Int J Med Microbiol 2001; 291 :57–62.11403412
8 Ghebremedhin B , KonigW, KonigB. Heterogeneity of methicillin-resistant Staphylococcus aureus strains at a German university hospital during a 1-year period. Eur J Clin Microbiol Infect Dis 2005; 24 :388–98.15931455
9 Monecke S , CoombsG, ShoreAC, et al A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One 2011; 6 :e17936.21494333
10 Effelsberg N , SteggerM, PeitzmannL, et al Global epidemiology and evolutionary history of Staphylococcus aureus ST45. J Clin Microbiol 2020; 59 :e02198-20.33087430
11 Moore CL , Osaki-KiyanP, PerriM, et al USA600 (ST45) methicillin-resistant Staphylococcus aureus bloodstream infections in urban Detroit. J Clin Microbiol 2010; 48 :2307–10.20335422
12 Sakoulas G , GuramK, ReyesK, NizetV, ZervosM. Human cathelicidin LL-37 resistance and increased daptomycin MIC in methicillin-resistant Staphylococcus aureus strain USA600 (ST45) are associated with increased mortality in a hospital setting. J Clin Microbiol 2014; 52 :2172–4.24648548
13 King JM , KulhankovaK, StachCS, VuBG, Salgado-PabonW. Phenotypes and virulence among Staphylococcus aureus USA100, USA200, USA300, USA400, and USA600 clonal lineages. mSphere 2016; 1 :e00071-16.27303750
14 O’Brien FG , CoombsGW, PearmanJW, et al Population dynamics of methicillin-susceptible and -resistant Staphylococcus aureus in remote communities. J Antimicrob Chemother 2009; 64 :684–93.19713400
15 Lee YT , LinDB, WangWY, et al First identification of methicillin-resistant Staphylococcus aureus MLST types ST5 and ST45 and SCCmec types IV and Vt by multiplex PCR during an outbreak in a respiratory care ward in central Taiwan. Diagn Microbiol Infect Dis 2011; 70 :175–82.21596221
16 Tsao FY , KouHW, HuangYC. Dissemination of methicillin-resistant Staphylococcus aureus sequence type 45 among nursing home residents and staff in Taiwan. Clin Microbiol Infect 2015; 21 :451–8.25677257
17 Wu TH , LeeCY, YangHJ, et al Prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus among nasal carriage strains isolated from emergency department patients and healthcare workers in central Taiwan. J Microbiol Immunol Infect 2019; 52 :248–54.30292763
18 Liu CY , LaiCC, ChiangHT, et al Predominance of methicillin-resistant Staphylococcus aureus in the residents and environments of long-term care facilities in Taiwan. J Microbiol Immunol Infect 2019; 52 :62–74.29530709
19 Lee CY , FangYP, WuTH, ChangYF, SungCH. Sequence types 8, 59, and 45 methicillin resistant Staphylococcus aureus as the predominant strains causing skin and soft tissue infections in Taiwan's prisons and jails. J Microbiol Immunol Infect 2022; 55 :1239–45.34635424
20 Huang YC , ChenCJ, LauderdaleTY. Detection, spread and phylogeny of meticillin-resistant Staphylococcus aureus sequence type 45 in Taiwan. Microb Genom 2021; 7 :000555.33843577
21 Lai CC , LeeCM, ChiangHT, et al Methicillin-resistant Staphylococcus aureus sequence type 45 with high rates of ciprofloxacin and tetracycline resistance in the residents and environments of long-term care facilities in Taiwan. J Infect 2018; 76 :305–7.29179975
22 Peng KT , HuangTY, ChiangYC, et al Comparison of methicillin-resistant Staphylococcus aureus isolates from cellulitis and from osteomyelitis in a Taiwan hospital, 2016–2018. J Clin Med 2019; 8 :816.31181643
23 Stepanovic S , VukovicD, DakicI, SavicB, Svabic-VlahovicM. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 2000; 40 :175–9.10699673
24 Peng KT , HsiehCC, HuangTY, et al Staphylococcus aureus biofilm elicits the expansion, activation and polarization of myeloid-derived suppressor cells in vivo and in vitro. PLoS One 2017; 12 :e0183271.28813499
25 Hooper DC , JacobyGA. Topoisomerase inhibitors: fluoroquinolone mechanisms of action and resistance. Cold Spring Harb Perspect Med 2016; 6 :a025320.27449972
26 Piddock LJ , JinYF, WebberMA, EverettMJ. Novel ciprofloxacin-resistant, nalidixic acid–susceptible mutant of Staphylococcus aureus. Antimicrob Agents Chemother 2002; 46 :2276–8.12069989
27 Campion JJ , McNamaraPJ, EvansME. Evolution of ciprofloxacin-resistant Staphylococcus aureus in in vitro pharmacokinetic environments. Antimicrob Agents Chemother 2004; 48 :4733–44.15561851
28 Masuda I , MatsubaraR, ChristianT, et al tRNA methylation is a global determinant of bacterial multi-drug resistance. Cell Syst 2019; 8 :302–14.e8.30981730
29 Vestergaard M , LengB, HaaberJ, BojerMS, VeggeCS, IngmerH. Genome-wide identification of antimicrobial intrinsic resistance determinants in Staphylococcus aureus. Front Microbiol 2016; 7 :2018.28066345
30 Hassanzadeh S , GanjlooS, PourmandMR, MashhadiR, GhazviniK. Epidemiology of efflux pumps genes mediating resistance among Staphylococcus aureus; a systematic review. Microb Pathog 2020; 139 :103850.31706002
31 Chen HJ , TsaiJC, HungWC, TsengSP, HsuehPR, TengLJ. Identification of fusB-mediated fusidic acid resistance islands in Staphylococcus epidermidis isolates. Antimicrob Agents Chemother 2011; 55 :5842–9.21968364
32 Zhao H , WangX, WangB, et al The prevalence and determinants of fusidic acid resistance among methicillin-resistant Staphylococcus aureus clinical isolates in China. Front Med (Lausanne) 2021; 8 :761894.34917634
33 Mlynarczyk-Bonikowska B , KowalewskiC, Krolak-UlinskaA, MaruszaW. Molecular mechanisms of drug resistance in Staphylococcus aureus. Int J Mol Sci 2022; 23 :8088.35897667
34 Kozitskaya S , ChoSH, DietrichK, MarreR, NaberK, ZiebuhrW. The bacterial insertion sequence element IS256 occurs preferentially in nosocomial Staphylococcus epidermidis isolates: association with biofilm formation and resistance to aminoglycosides. Infect Immun 2004; 72 :1210–5.14742578
35 Loessner I , DietrichK, DittrichD, HackerJ, ZiebuhrW. Transposase-dependent formation of circular IS256 derivatives in Staphylococcus epidermidis and Staphylococcus aureus. J Bacteriol 2002; 184 :4709–14.12169594
36 Wan TW , KhokhlovaOE, IwaoY, et al Complete circular genome sequence of successful ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (OC8) in Russia: one-megabase genomic inversion, IS256’s spread, and evolution of Russia ST8-IV. PLoS One 2016; 11 :e0164168.27741255
37 Kleinert F , KalliesR, HortM, et al Influence of IS256 on genome variability and formation of small-colony variants in Staphylococcus aureus. Antimicrob Agents Chemother 2017; 61 :e00144-17.28584147
38 Kuroda M , SekizukaT, MatsuiH, OhsugaJ, OhshimaT, HanakiH. IS256-mediated overexpression of the WalKR two-component system regulon contributes to reduced vancomycin susceptibility in a Staphylococcus aureus clinical isolate. Front Microbiol 2019; 10 :1882.31474962
