Genetic Diversity of Methicillin-Resistant Staphylococcus Aureus Among Japanese Inpatients in the Early 1980s

Background: Methicillin-resistant Staphylococcus aureus (MRSA) was recognized as a serious nosocomial pathogen in Japan starting in the early 1980s. Limited genotypic characteristics on healthcare-associated MRSA (HA-MRSA) associated with “hospital MRSA panics” in Japan in this era. Therefore, this study aimed to determine the characteristics of Japanese HA-MRSA strains in the early 1980s and their linkage with current MRSA strains. Methods: For 194 S. aureus strains isolated in the early 1980s, we determined methicillin resistance phenotypically and genotypically and performed multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and whole-genome sequencing. Results: Twenty mecA-positive MRSA (10.3%), 8 mecA-negative MRSA (4.1%) and 3 mecA-positive methicillin-susceptible S. aureus (MSSA) (1.5%) strains were identied. The frequent sequence type (ST) was ST30 (n=11), followed by ST5 (n=8), ST81 (n=4), and ST247 (n=3). Rates of SCCmec types I, II, and IV were 65.2%, 13.0%, and 17.4%, respectively. ST30-SCCmec I (n=7) and ST5-SCCmec I (n=5) were predominant genotypes. Only two strains exhibited tst-positive ST5-SCCmec II, which is the current Japanese HA-MRSA genotype. Moreover, 73.3% of SCCmec type I strains were susceptible to imipenem compared with SCCmec type II strains (0%). All ST30 strains shared a common ancestor with strain 55/2053, which resulted in a global pandemic of Panto-Valentine leukocidin (PVL)-positive and penicillin-resistant MSSA spread in Europe and the United States in the 1950s. Conclusions: Our results demonstrated the heterogeneous population structure of Japanese HA-MRSA early which comprised diverse clones rare


Background
Staphylococcus aureus is a major opportunistic pathogen that can cause various life-threatening infections, and approximately 20% of healthy human individuals are persistent carriers of this bacterial species [1]. The rst case of methicillin-resistant S. aureus (MRSA) was identi ed in the United Kingdom in 1961, just in one year after introduction of methicillin [2,3]. Since then, MRSA remains a major clinical concern with both hospital-associated and community-associated MRSA (HA-MRSA and CA-MRSA, respectively) infections worldwide [4][5][6]. The number of deaths related to MRSA infection is still large and rivals HIV/AIDS infection in its public health impact [7].
Nasal carriage of bacteria including S. aureus in human hosts can serve as a reservoir for the spread of the pathogen and can predispose the host to subsequent infections [8][9][10][11]. MRSA infections are often resistant to empirical antimicrobial therapy because of multidrug-resistance including β-lactam antimicrobials [12,13]. Therefore, nasal swabbing has been performed for MRSA screening as an infection control measure [14,15].
MRSA is generated when methicillin-susceptible S. aureus (MSSA) acquires the exogenous mecA gene encoding the penicillin-binding protein 2′ (PBP2′), which is located on a mobile genetic element named as staphylococcal cassette chromosome mec (SCCmec) [16]. According to the International Working Group on the Classi cation of Staphylococcal Cassette Chromosome Elements, strains can be classi ed by SCCmec types (I-XIV) based on the combinations of mec and ccr gene complexes [6,[17][18][19]. Each MRSA clone has been described by the combination of the chromosomal genotype of the recipient MSSA strain and the genotype of the integrated SCCmec. Therefore, multilocus sequence typing (MLST) and SCCmec typing by PCR and/or sequencing methods have been widely used as gold standard methods in molecular epidemiological studies of MRSA [4,[20][21][22][23]. In addition, whole-genome sequencing (WGS) of bacterial isolates by next-generation sequencing (NGS) technologies has recently become a promising tool for molecular typing. WGS by NGS has made remarkable advances for microbiologists; these changes re ect decreased costs, increased capabilities of data processing systems, and ever-larger databases of NGS datasets [24]. However, these molecular typing tools for MRSA isolates were established in the 2000s; thus, there have been few reports on molecular epidemiology of MRSA strains before then.
Population structure tracking of bacteria over time can provide crucial insights into infection control and appropriate use of antimicrobials in clinical practice [25]. Therefore, the aims of this study were to retrospectively review the population structure of Japanese HA-MRSA strains isolated in the early 1980s using MLST, SCCmec typing and phylogenetic analysis based on whole-genome single nucleotide polymorphisms (SNPs), and to compare the population structure with that of strains isolated in recent years. Consequently, we report the polyclonal population structure of Japanese HA-MRSA in the 1980s, which were composed of rare clones in recent years.

DNA extraction
A single colony of each strain was used for bacterial genomic DNA extraction by using the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany). Puri ed genomic DNA was used for PCRs and sequencingbased methods.

Determination of methicillin-resistance
We determined methicillin-resistance in all S. aureus strains by evaluating oxacillin and cefoxitin susceptibilities according to Clinical and Laboratory Standards Institute (CLSI) M100-S22 performance standards [28]. In addition, all strains were genetically assessed by two different PCRs for the presence of the mecA gene [29,30].

WGS and additional molecular phylogenetic analysis
The Nextera XT DNA sample preparation kit (Illumina Inc., San Diego, CA, USA) was used for sample preparation for WGS. Sequencing was performed using a paired-end 2 × 250 or 300-bp cycle run on the Illumina MiSeq sequencing system using MiSeq reagent kit v2 or v3 (Illumina Inc.).
Assembled contigs of strains isolated in this study were also submitted to CSI phylogeny 1.4 on the CGE website to call SNPs and infer the phylogenetic relationship among strains described in this study and the 125 complete genome sequenced-S. aureus strains, which were available from the National Center for Biochemistry Information (NCBI) database [40]. In addition, pairwise SNP analyses were performed focusing on clonal complexes (CC) 5 and CC30 strains in order to elucidate relatedness with and preservation among the recent MRSA strains.

Results
Rates of MRSA and MSSA strains in Japanese hospitals during the early 1980s In total, 194 isolates were identi ed as S. aureus. One hundred and seventy-four patients yielded one strain per each patient, whereas ten patients yielded two strains per each patient. According to phenotypic and genotypic determination of methicillin-resistance, we identi ed 20 mecA-positive MRSA (10.3%), 8 mecA-negative MRSA (4.1%), 3 mecA-positive MSSA (1.5%) and 163 mecA-negative MSSA (84.0%) strains. Consequently, the phenotypic methicillin-resistant rate of the strains described in this study was 14.4% (28 of 194 strains).
Antimicrobial susceptibilities in MRSA and MSSA strains from Japanese inpatients during the early 1980s As shown in Table 1, all S. aureus strains described in this study were susceptible to levo oxacin, which had been not launched in Japan in the 1980s. All S. aureus strains were susceptible to anti-MRSA agents including vancomycin, teicoplanin, linezolid, and arbekacin, irrespective of methicillin resistance.
Moreover, 32.1% of MRSA strains (9 of 28 strains) showed resistance to imipenem, which was unavailable in Japan during the study period, whereas all MSSA strains were susceptible to imipenem.
Sulfamethoxazole/ trimethoprim Detailed genetic characterizations of MRSA and mecA-positive MSSA strains in Japan during the early 1980s In order to understand the detailed characteristics of Japanese HA-MRSA strains during the early 1980s, we performed genetic characterizations using MLST, SCCmec typing, spa-typing, toxin pro ling and acquired antimicrobial resistance gene pro ling ( Table 2, Additional_ le_2).
Consequently, the prevalence of various MRSA clones during the early 1980s was found in this study, and diverse genotypes were detected even among MRSA strains exhibiting the same ST. These results suggested that the population structure of Japanese HA-MRSA strains during the early 1980s had been notably different from the monoclonal population composed of ST5-SCCmec II known as HA-MRSA in recent years.

Antibiograms of the strains by SCC mec types
In order to consider the mechanisms for shift of population structure of Japanese HA-MRSA strains from polyclonal during the early 1980s to monoclonal in recent years, we compared the antibiograms of MRSA strains across SCCmec types (Table 3, Additional_ le_3).
Strains that carried SCCmec types I and II were highly resistant to β-lactams including oxacillin, however, those that carried SCCmec type IV were more susceptible to β-lactams despite being mecA-positive. Among 15 strains carrying SCCmec type I, the rate of erythromycin resistance was the highest (80.0%), followed by resistance to gentamycin (66.7%), clindamycin (40.0%), and minocycline (26.7%), which were commonly used antimicrobials at that time. Imipenem susceptibility rates in SCCmec type I strains were 73.3% (11 of 15 strains) as compared with 0% in SCCmec type II strains (0 of 3 strains) (p < 0.05).
The mecA-negative MRSA strains (n = 8) were more susceptible to β-lactams, and especially, all strains were susceptible to imipenem except one isolate (N89). Lower MICs of these strains for aminoglycosides, minocycline, erythromycin and clindamycin were observed compared with the mecA-positive strains.
Population structure of MRSA and mecA-positive MSSA strains in Japan during the early 1980s In order to understand the population structure of Japanese HA-MRSA strains during the 1980s, we performed a phylogenetic analysis based on whole-genome SNPs for 20 mecA-positive MRSA, 8 mecAnegative MRSA, 3 mecA-positive MSSA, and 125 reference S. aureus strains (Fig. 1).
The neighbor-joining (NJ) tree was constructed by alignment of 41,910 SNP sites. The percentage of reference genome (Staphylococcus argenteus MSHR1132) covered by all isolates was 89.27% (1,739,038 of 2,762,785 positions). Strains belonging to the CC30 cluster were most predominant (n = 11), followed by CC5 (n = 9), CC1 (n = 4), and CC8 (n = 4), indicating that the population structure of MRSA strains during the early 1980s was composed of diverse clones.

Whole-genome SNP analysis of CC30 and CC5 strains
In order to elucidate whether prevalence of MRSA strains in this study resulted from a recent transmission event, the detailed pairwise SNPs analyses in 11 ST30 and 9 CC5 strains described in this study were performed, respectively ( Figs. 2A and 2B).
The detailed pairwise SNPs analysis of 11 ST30 strains described in this study and 6 CC30 reference strains was performed ( Fig. 2A). The percentage of reference genome (SJTUF_J27, ST433) covered by all strains was 89.16% (2,500,756 of 2,804,761 positions) in the SNPs analysis. SNP differences ranged from 28 to 1008. All CC30 strains described in this study clustered into a single clade and were most closely related to the MSSA strain 55/2053 isolated in the United Kingdom in 1955 [41]. N83 and N86 strains, which were isolated in Kumamoto in the same year and exhibited the same spa-type t1504, showed 28 SNP differences, suggesting a direct horizontal spread in this hospital. However, SCCmec type and antimicrobial resistance gene pro les differed between these two strains, suggesting that these strains were independently acquired by each inpatient from different infectious sources. Consequently, the ST30 strains isolated in this study could be recognized as causing a regional outbreak, with the number of SNPs ranging from 28 to 164 [42,43].
Next, we performed SNP analysis among 9 CC5 MRSA strains in this study and 32 reference strains (Fig. 2B). The percentage of reference genome (ED98) covered by all strains was 89.13% (2,517,393 of 2,824,404 positions) in the SNP analysis. SNP differences ranged from 0 to 621. ST5 MRSA strains described in this study clustered into ve different clades. Strains N366, N98, N345, N279, and N283 harboring SCCmec type I belonged to a single cluster, and SNP differences ranged from 30 to 84. N279 and N283 strains, which were isolated in Miyagi in the same year, showed only 30 SNP differences and similar pro les regarding spa type and toxin and antimicrobial resistance genes, suggesting a recent transmission event. Although N366 and N98 strains only exhibited 41 SNP differences, these strains were isolated in a different cross-strait region ( Table 2, Additonal_ le_2). According to these results, strains exhibiting ST5-SCCmec I belonging to this clade could be recognized as causing a regional outbreak, as contrasted with strains 16125, 18412, 18341, 10388, 15532, 18583, 16035, and 10497, which were recovered with the number of SNPs ranging from 0 to 7 at a hospital during the outbreaks in Switzerland (Fig. 2B) [44].
Thus, our results suggested that MRSA clones exhibiting ST30-and ST5-SCCmec I had been spread as regional clones throughout Japan in the early 1980s.

Discussion
This study indicated that the population of Japanese HA-MRSA strains present during the early 1980s showed a polyclonal structure composed of multiple clones, most of which have become uncommon recently. The recent monoclonal population structure of HA-MRSA strains in Japan likely formed over the past several decades, possibly in response to the release of various new antimicrobial agents and changes in MRSA treatment strategies from the 1980s onward.
This study indicated that PVL-positive ST30-SCCmec I was the most frequent genotype among Japanese HA-MRSA strains in the early 1980s. According to a previous report, nosocomial outbreaks of MRSA exhibiting PVL-positive ST30-SCCmec IV had frequently occurred in the late 1980s and early 1990s in Japanese hospitals [45], whereas this genotype comprised a minor population in the early 1980s in this study. Our results suggested that the population structure of Japanese HA-MRSA strains had been dynamically replaced through the 1980s. This dynamic replacement largely due to ST30 MRSA clones might have resulted from high genetic and phenotypic diversity among ST30 MRSA strains. Indeed, in addition to mecA-positive MRSA, mecA-negative MRSA and mecA-positive MSSA, which were rarely isolated in recent years, were also found among ST30 strains in this study. Differences in observed SCCmec types and presence or absence of PVL genes were also identi ed among these strains.
As previously reported, the CC30 S. aureus lineage can be divided into three clusters: Clade 1 (representative strain 55/2053; PVL-positive and penicillin-resistant MSSA), Clade 2 (representative strain TCH60; PVL-positive CA-MRSA harboring SCCmec type IV), and Clade 3 (representative strain MRSA252/EMRSA-16; PVL-negative HA-MRSA harboring SCCmec type II or IV) [41]. Strains in Clade 1 cause severe infections and had been the epidemic clone in Europe, the United States, and Australia in the 1950s [46][47][48][49][50]. The percent of S. aureus infections caused by Clade 1 strains decreased dramatically by the mid-1960s, resulting from methicillin use for the treatment of penicillin-resistant strains [51]. However, this clone had re-emerged as HA-MRSA in Japanese hospitals in the early 1980s according to our results. Our phylogenetic analysis based on whole-genome SNPs demonstrated that all Japanese ST30 strains clustered into a single clade including strain 55/2053, suggesting that a Clade 1 strain imported from overseas had acquired SCCmec type I, SCCmec type IV, or unknown genetic factors and had already increased diversity in Japanese hospitals in the early 1980s. Consequently, the ST30 strains had likely spread throughout Japan as a nosocomial clone causing a regional outbreak at that time.
We previously reported that strains harboring SCCmec type II accounted for a large portion of MRSA in Japanese hospitals in the late 1990s [20,52]. The polyclonal population structure of MRSA strains in the early 1980s observed in this study had been replaced by a monoclonal population structure largely composed of the current Japanese HA-MRSA clone since the late 1990s, coinciding with the new release of various antimicrobial agents such as third-generation cephalosporins, carbapenems and uoroquinolones from the 1980s onward. The treatment of MRSA infections had been dramatically altered at those times. Imipenem was frequently used for the treatment of MRSA infections before the clinical introduction of vancomycin in 1991 in Japan. Strains harboring SCCmec type I, which was the predominant genotype in this study and some countries including the United Kingdom in the early 1980s [20,53], displayed high rate of imipenem-susceptibility, as contrasted with SCCmec II strains. Imipenem/cilastatin was launched in 1987 and had been used as an anti-MRSA agent before the clinical introduction of vancomycin in 1991 in Japan. According to the data in Japanese hospitals, the simple selective pressure induced by imipenem, might have caused dynamic population shift from the imipenem-susceptible diverse MRSA clones to the imipenem-resistant ST5-SCCmec II MRSA between 1980 and 2000. Also, in vitro exposure to imipenem can induce the conversions of heterogeneous-tohomogeneous and Eagle type-to-homogeneous methicillin resistance in S. aureus strains via chromosomal gene mutations such as vraSR and rpoB genes [6,21,[54][55][56][57][58]. Thus, the clinical use of imipenem against MRSA infections might have contributed to the attenuation of MRSA strains with atypical methicillin-resistance phenotype, which were rarely isolated in recent years.
In the current study, multiple MRSA strains exhibiting ST247-SCCmec I were isolated in the northeast area in Japan. This genotype is known as the Iberian clone, and had been dominated internationally up to the early 2000s [59][60][61][62]. Our results showed the local existence of the Iberian clone in Japan during the early 1980s. Interestingly, all of ST247-SCCmec I strains in this study were resistant to imipenem. At the start of clinical use of imipenem around the world, the Iberian clone might have been spread from the 1990s to the early 2000s. The Iberian clone has been already replaced by the current major epidemic clones [63].
This study showed that ST5-SCCmec I was the second most frequent genotype among Japanese HA-MRSA strains in the early 1980s. This genotype is shared by EMRSA-3, which was the most common MRSA clone in the United Kingdom in 1987-1988 along with EMRSA-15 (ST22-SCCmec IV) and EMRSA-16 (ST36-SCCmec II) [4]. Studies conducted in South America in the late 1990s have identi ed the Cordobes/Chilean clone, which is genetically related to the EMRSA-3 but presents differences in the pulsed-eld gel electrophoresis (PFGE) pattern and spa type [64][65][66]. This MRSA clone was also detected at a high rate in hospitals in South Brazil in 2008, suggesting the potential for re-dissemination in Brazil [67,68]. Although this MRSA clone exhibiting ST5-SCCmec I has remained uncommon in regions outside of South America in recent years, continuous monitoring is needed to prevent future outbreaks.
In 2001, we reported on the complete genome sequence of the S. aureus strain N315 with ST5-SCCmec II, as a representative strain of New York/Japan HA-MRSA clone [69]. The strain N315 was isolated from the pharyngeal smear of a Japanese patient in 1982, and also described in this study. Our results showed that the Japanese HA-MRSA clone exhibiting genotype of the strain N315 with multi-drug resistance had already been existed as a minor clone in the early 1980s, and could be the most frequent HA-MRSA in the 1990s and 2000s due to wide use of new antimicrobials. Whereas in the 2010s, awareness of appropriate use of antimicrobials has been raised to combat antimicrobial resistance issues in the world. It was reported that the population of Japanese HA-MRSA had been shifting again from N315-like CC5-SCCmec II to CC8-SCCmec IV and CC1-SCCmec IV, both of which had higher susceptibility to cefotaxime, levo oxacin, clarithromycin and clindamycin in the 2010s [70]. The history of the clonal change of MRSA in decades seems to re ect the recent appropriateness of our antimicrobial usage.

Conclusions
In conclusion, this study revealed the alteration of population structures of HA-MRSA strains from the early 1980s onward as a consequence of elimination by new drug developments over the past several decades. Our ndings will contribute to future progress of infection control measurements and appropriate use of antimicrobials for nosocomial infections caused by MRSA.

Consent for publication
Not applicable.

Availability of data and materials
The read data for whole-genome sequencing analysis of strains in this study have been deposited in GenBank under accession number DRA010146. Other data and materials in this study are included within the article.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by a Grant-in-Aid (S1201013) from the Ministry of Education, Culture, Sports, Science, and Technology Supported Program for the Strategic Research Foundation at Private Universities (2012-2016). The funder had no role in study design, data collection, and interpretation or the decision to submit the work for publication.
Authors' contributions HZ, YU and KH participated in the study design; HZ, YU and KH collected and analyzed clinical information; HZ, YU, YL, TS and KH carried out all microbiological experiments and data analysis; HZ, YU, TS and KH drafted and revised the manuscript. All authors read and approved the nal manuscript.
Explanation of the additional les Additional_ le_2.xls.  Figure 1 Phylogenetic tree of the strains in this study and other reference genomes. Phylogenetic tree based on whole-genome SNPs of reference genomes (black-colored font) including mecA-positive MRSA (redcolored font), mecA-negative MRSA (blue-colored font), and mecA-positive MSSA (green-colored font) isolated in this study. NJ tree was constructed by alignment of 41,910 SNP sites. Staphylococcus argenteus MSHR1132 was used as the outgroup. CC, clonal complex; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus; NJ, neighbor-joining; SNPs, single nucleotide polymorphisms; ST, sequence type.