Molecular Characterization of Methicillin-Resistant Staphylococcus aureus from Outpatients in Northern Japan: Increasing Tendency of ST5/ST764 MRSA-IIa with Arginine Catabolic Mobile Element

Introduction

Staphylococcus aureus is a major human pathogen that causes a wide spectrum of diseases, ranging from superficial skin and soft tissue infections to life-threatening septicemia, endocarditis, and toxic shock syndrome, and it possesses remarkable ability to acquire resistance to newer antimicrobials. Methicillin-resistant S. aureus (MRSA) is the most leading cause of healthcare- as well as community-associated infections worldwide. ¹ Methicillin resistance of MRSA is conferred by mecA gene, which encodes an alternative penicillin-binding protein (PBP2a) with a low affinity to β-lactams, and it is carried within a transmissible genome element, staphylococcal cassette chromosome mec (SCCmec). The SCCmec in MRSA is highly diverse in structural organization and genetic content, and has been classified into at least 12 SCCmec types (I–XII),^2–4 among which types I, II, and III are commonly detected in healthcare-associated MRSA (HA-MRSA); whereas types IV and V are frequently reported in community-associated MRSA (CA-MRSA). ⁵ However, several HA-MRSA clones (e.g., EMRSA-15) also carry SCCmec IV, and genetic distinction between HA-MRSA and CA-MRSA has currently become obscure. ⁶ Panton–Valentine leukocidin (PVL), a prophage-encoded bicomponent pore-forming protein, is prevalent in a part of CA-MRSA strains and is associated with increased virulence of S. aureus, leading to severe skin and soft tissue infections and necrotizing pneumonia. ⁵

Among the CA-MRSA, the USA300 clone (PVL-positive ST8 MRSA-SCCmec IV) is currently the most commonly distributed in the United States and has been spreading worldwide.^7,8 USA300 clone is characterized by the presence of arginine catabolic mobile element (ACME, type I), which probably originated from S. epidermidis, a genomic island juxtaposed to the SCCmec IV that contains arc cluster, opp-3 cluster, and speG. ⁹ The arc cluster mediates the arginine-deiminase pathway that converts L-arginine to L-ornithine for production of both ammonia and ATP.^9,10 Hence, the bacterial cells are considered to become tolerant to the acidic environment of human skin and acquire enhanced fitness to the host. The speG encodes a spermine/spermidine N-acetyltransferase (SpeG), which detoxifies polyamines that are bactericidal substances produced by all living organisms.^11,12 SpeG allows the USA300 strains to evade the toxicity of polyamines secreted on the human skin and to potentiate its colonization and infection. Another type of ACME, ACME II, which lacks opp-3 cluster that encodes oligopeptide permease operon, was reported in S. epidermidis.^9,13 The truncated form ACME II, that is, ACME II′ (previously referred to as ΔACME II) has also been detected in S. epidermidis ¹⁴ and MRSA with various lineages: ST5-SCCmec II/IV/V, ST8-SCCmec IVa, ST22-SCCmec IVh, ST239-SCCmec III, ST398-SCCmec IV, and ST764-SCCmec II.^15–25 Similar to ACME I, ACME II′ is predicted to enhance survival and growth of MRSA in the host.^16,17,22 Accordingly, although the prevalence of ACME II′ among MRSA is low at present, the spread of MRSA harboring ACME II′ is considered a potential public health concern and needs to be monitored in both healthcare settings and the community.

In Japan, the most prevalent MRSA clone is ST5-MRSA-SCCmec II, which is known as New York/Japan clone, ²⁶ whereas USA300 has been rarely detected. In our previous epidemiological study in 2009 of CA-MRSA in Hokkaido, northern main island, prevalence rate of MRSA was 18.6% among S. aureus isolates from outpatients, and ACME II′ was identified in an ST5-MRSA-SCCmec II (strain SR388) and ST5-MRSA-SCCmec V (strain SR141). ²⁷ Further genetic analysis revealed that these strains possessed novel genetic structures of ACME-SCCmec composite island (CI) distinct from that of the USA300 clone. ²⁴ Thereafter, ACME II′ was detected in several MRSA isolates with ST5 and ST764 from outpatients as well as hospitalized patients until 2011, and five genetic variants of ACME-SCCmec CI were identified among these isolates. ²⁸ Similarly, ST764 MRSA having ACME II′ was also reported in another place in Japan. ²³

The present study was conducted in 2013–2014 in Hokkaido, Japan, to investigate genetic characteristics of MRSA isolated from outpatients after the preceding studies. We report here an increasing trend of ACME II′-harboring ST5/ST764 MRSA-SCCmec II and identification of new genetic variations in ACME-II′-SCCmec CI.

Materials and Methods

Results

Among a total of 624 MRSA isolates, both PVL genes and ACME were detected in 9 isolates (1.4%), whereas solely PVL genes or ACME were detected in 2 (0.3%) and 36 isolates (5.8%), respectively (Table 1). Most of the isolates belonged to coagulase (coa) genotype IIa (455, 72.9%), followed by IIIa (74, 11.9%), VIIa (51, 8.2%), Ia (17, 2.7%), and Va (16, 2.6%). The dominant SCCmec type was IIa (439, 70.4%) followed by IVa (78, 12.5%), V (34, 5.4%) IVl (24, 3.8%), and IVh (13, 2.1%). The 9 PVL- and ACME-positive isolates belonged to coa-IIIa, SCCmec IVa, and ACME type I, whereas all the 36 isolates with solely ACME were classified into coa-IIa, ACME II′, having SCCmec IIa (32 isolates) or V (4 isolates). The detection rate of ACME II′ among SCCmec II-MRSA was 7.1% (32/452): 5.5% (17/309) in 2013 (July to December) and 10.5% (15/143) in 2014 (January to March). These were found to be significantly increased rates (p < 0.0001) as compared with those in 2009 (0.86%) and 2011 (4.5%). ²⁸

Further genetic analysis was performed for a total of 31 strains comprising PVL⁺/ACME I (9), PVL⁺/ACME⁻ (2), PVL⁻/ACME II′ (15), and PVL⁻/ACME⁻ (5) MRSA. These strains included all the 11 PVL-positive isolates. Among the 36 PVL⁻/ACME II′ isolates, 15 isolates derived from patients with different age/sex and various clinical specimens were selected (Tables 2 and 3). The 5 PVL⁻/ACME⁻ strains were chosen randomly from the 24 MRSA having SCCmec IVl. All the PVL⁺/ACME I strains had PVL prophage ΦSa2usa and belonged to ST8, spa type t008, agr group I, exhibiting typical traits of USA300 clone,^5,9 and were derived from mostly pus, skin, or wound. Two PVL⁺/ACME⁻ strains with SCCmec IVc and V were isolated from pus and carried ΦPVL, and they belonged to ST30/agr-III/coa-IVa and ST59/agr-I/coa-Va, respectively. 15 PVL⁻/ACME II′ MRSA strains were classified into ST5-SCCmec IIa or V (7 strains), and ST764-SCCmec IIa (8 strains), and five spa types with t002 being dominant. PVL⁻/ACME⁻ MRSA strains with SCCmec IVl were assigned to ST8 and spa-t1767 or t5071.

Compared with PVL⁺/ACME I ST8-MRSA having SCCmec IVa, PVL⁻/ACME II′ ST5 and ST764-MRSA-SCCmec IIa strains were resistant to more antimicrobials, including gentamicin, fosfomycin, and clindamycin, harboring more resistance genes to aminoglycosides and macrolides (Table 3). By sequencing analysis, seb and sec detected among the 31 strains were assigned into two subtypes (seb1, seb2) and sec3, respectively. Sequences of tst-1 detected in the present study were identical to those previously reported (e.g., GenBank accession no. AB084255). The ST5 MRSA with ACME II′ possessed mostly enterotoxin gene clusters (seg-sei-sem-sen-seo-seu) and some more enterotoxin genes (seb1, seb2, sec3, sel, sep). ST8-MRSA-SCCmec IVa harbored sek, seq, sak, and speG. bbp (bone sialoprotein gene) and cna (collagen binding protein gene) were detected only in PVL-positive ST30-MRSA-SCCmec IVc. All the ST764 ACME-II′ strains showed resistance to 13 antimicrobials. In contrast, ST5 ACME-II′ strains showed variable drug resistance patterns (resistant to 5–13 antimicrobials). seb2 was detected in the ST764 strains, whereas seb1 was detected in ST5 strains. speG was not detected in ST764, although 5 among 7 ST5 ACME-II′ strains harbored this gene. sasL was detected in all the 24 MRSA-SCCmec IVl isolates (data not shown), among which the five selected strains were identified in ST8. They were susceptible to most antimicrobials tested, harboring ant(4′)-Ia and mostly sec3 and tst-1, whose characteristics were different from PVL-positive ST8-MRSA-IVa.

Genetic organization of orfX, ACME, and SCCmec (ACME-SCCmec CI) of the 36 ACME II′-positive MRSA-SCCmec II or V was classified into five types (ii)–(vi) (Table 4). The dominant type was (ii) (25 isolates), which represents an ACME-SCCmec CI orfX-ΨSCC_{ΔJ1 SCCmec I}-ACME II′_SR388-SCCmec II, as identified for strain SR388 in our previous study. ²⁴ Among the three variants A1–A3 of this CI in terms of differences in the J3 region in SCCmec II, ²⁸ A3 was the most common (14 isolates) followed by A2 (11 isolates). Type (iii) orientation, which lacks ΨSCC_ΔJ1 in type (ii) (variant A2), was found in five isolates. Three isolates with SCCmec V had the type (iv) orientation, which is the same CI as previously identified for strain SR141. ²⁴ Although the location in the SCCmec-ACME could not be specified, speG was positive in two of the three SCCmec V isolates. The remaining two MRSA-SCCmec IIa (strain SC640, SC792) and MRSA-SCCmec V (strain SC995) had putative novel ACME-SCCmec CIs with type (v) and (vi) orientations, respectively. In these two CIs, SCC4610- or SCC266-like genetic elements ⁴² that contain speG were located adjacent to ACME II′. The SCCmec II in the CI type (v) was found to be the same as that described in A1 variant of type (ii) that contains additional ccrC1. The genetic organization of CI (ii)-variant A3 was detected in only ST764 strains, whereas CI (ii)-variant A2, (iv), (v), and (vi) were found in ST5-MRSA with different spa types (Table 2).

Discussion

In our previous studies on CA-MRSA in Hokkaido,^18,19,27 northern main island of Japan, we identified isolates with ACME-II′ in only ST5 MRSA in 2009, ²⁷ and ST5 and ST764 in 2011. ¹⁸ These findings suggested that ST5/ST764 clones with ACME-II′ have been emerging among CA-MRSA in northern Japan, indicating the importance to analyze prevalence and genetic characteristics of ACME-positive MRSA. The present study revealed that ACME-II′ has been increasing among ST5/ST764-SCCmecII MRSA strains. Notably, two novel SCCmec-ACME CIs carrying speG were identified in ST5 MRSA, in addition to the five SCCmec-ACME CIs previously described in northern Japan. ²⁸ Although speG had been found in ACME-I of USA300 clone,^9,12 speG was first identified in ST5 MRSA in the present study. Furthermore, the presence of PVL-negative ST8 MRSA having SCCmec IVl ³⁴ was first documented in Hokkaido, Japan. These findings suggested a changing epidemiology of MRSA, that is, spread of clones that are more adapted to the community in northern Japan, associated with genetic evolution.

In the United States, the ST8-MRSA-SCCmec IVa (USA300 clone) was presumed to have emerged as a community-associated pathogen during the early 1990s. This clone has been overwhelming among both CA- and HA-MRSA in the 2000s, replacing the previously prevalent ST5-MRSA-SCCmec II (New York/Japan clone).^8,43 Accordingly, this clone was recognized in Canada, South America, Europe, and Australia^5,7,44; thus, further spread to Asia was a matter of concern. However, in Japan, as well as other Asian countries, prevalence of ST8-MRSA-IVa has been still low since its emergence in the late 2000s.^{18,19,45–47} In our study in Hokkaido, ST8-MRSA-IV was detected in 1.1% (5/422) of MRSA from outpatients in 2011, ¹⁸ and in 0.3% (2/601) of MRSA isolates in a tertiary hospital during 2008–2010. ¹⁹ In the present study in 2013–2014, its detection rate was 1.4% (9/624) among isolates from outpatients. Therefore, prevalence of ST8-USA300 clone is considered to have been remaining at a low level.

In contrast, ACME-positive isolates among MRSA with SCCmec II increased remarkably, rising from 0.86% in 2008 ²⁸ to 10.5% in 2014 in Hokkaido, Japan. The ACME-positive MRSA consists of two clones, ST5 and ST764, as seen in the present and our previous studies.^18,19,27 ST764 is a single locus variant of ST5, and it has been prevalent in Japan, including Tokyo. ⁴⁸ Among the ST764-MRSA-SCCmec II, ACME-positive isolates were identified in our study in Hokkaido, ¹⁸ and also in Niigata ²³ located on the northwest coast of Honshu island in Japan. Although ST764-MRSA-SCCmec II was previously assigned only to spa-type t002,^18,23 another type t548, which is closely related to t002, was detected in the present study. Similarly, two new spa-types (t045, t2487) were found in ST5-MRSA-SCCmec II (previously detected types: t002, t067, t071, and t3557). These findings suggested expansion of genetic diversity in ST5/ST764 MRSA with ACME, probably due to increased frequency of transmission among the population. This view may be also supported by some differences in the presence of drug resistance genes, virulence factors, and adhesion genes among these strains observed in the present study. In contrast, ST8-MRSA-SCCmec IVa strains were highly homogenous, suggesting low frequency of genomic evolution, probably due to less transmission frequency.

Although an increase of ACME among MRSA was noted in the present study, majority of CA-MRSA isolates from northern Japan investigated were PVL negative (98.2%) and ACME negative (92.8%). This finding is in agreement with studies from other countries.^25,47,49 Accordingly, it seems that PVL and/or ACME are not essential factors for MRSA to thrive in the community.

Among the ACME-SCCmec CIs identified in the present study, the A3 variant of type (ii) (orfX-ΨSCC_{ΔJ1 SCCmec I}-ACME II′_SR388-SCCmec II) was the most common, followed by the A2 variant of type (ii). In our previous study, only three isolates with A2 variant were detected in 2011, whereas the A3 variant was found in seven isolates. ²⁸ Thus, a potential increase of A3 variants is suggested, despite the low number of isolates assigned to A2 and A3 variants. Length of ACME-SCCmec CI-A3 variant (72 kb) is shorter than A2 variant by ∼4 kb, lacking pUB110 within SCCmec II, which may confer lower fitness cost to the host bacterium to which the CI is transferred horizontally, as previously speculated. ²⁸

It was of note that the two putative novel ACME-SCCmec CIs [types (v) and (vi)] that contain speG were detected. CI type (v) is considered a modified form of type (ii)-variant A1, in which SCC4610-like genetic element is inserted between ACME II′_SR388 and SCCmec II. Similar to the CI in ST5-MRSA-SCCmec V strain SR141 (orfX-ACME II′_SR141-SCCmec V), CI type (vi) has SCCmec V but is associated with ACME II′_SR388 (without IS1182), and it contains SCC266-like element located downstream from orfX. The SCC4610 and SCC266 were described in MRSA-SCCmec V strain JCSC4610 and MRSA-SCCmec II J266, respectively, ⁴² and they were located just downstream of orfX and contained speG at positions in proximity to orfX. SpeG, acetyltransferase for polyamines has been believed to be associated with only USA300 MRSA carrying ACME I, and to contribute to detoxify spermine that is overproduced by increased L-ornithine via arc cluster.^9,10 The presence of speG through acquisition of speG-carrying SCC, as found in ACME II′-SCCmec CI type (v) and (vi), is suggested to confer biological benefit on bacterium having arc cluster, by countering increased level of spermines,^10,50 as seen in ACME I. Further biological and epidemiological studies are necessary to determine the effect of SpeG to enhance virulence and/or colonization ability of ACME II′-positive S. aureus.

In our present study, except for ST8-MRSA-SCCmec IVa, one strain each of ST30 MRSA-SCCmec IVc and ST59 MRSA-SCCmec V were isolated as those harboring PVL genes. These are known as major CA-MRSA clones in Asia, particularly prevalent in Southeast Asia (ST30) and Taiwan (ST59), ²⁶ and were isolated in our previous studies^18,27 and other studies in Japan. ⁴⁶ Despite the still low frequency among CA-MRSA, these clones are considered to have been persisting in Japan. Remarkably, PVL-negative ST8 MRSA carrying a novel SCCmec IV subtype, SCCmec IVl ³⁴ was identified in the present study. The ST8-MRSA-SCCmec IVl was designated ST8 CA-MRSA/J since its description in 2003. This MRSA clone is characterized as spa-type t1767, agr-I, coa-III, harboring mostly tst-1, sec, sel, and sep, and it has a novel cell wall-anchored surface protein (SasL or CWASP/W) encoded by sasL (spj) located in the J1 region of the SCCmec IVl.^34,35,51 The ST8 CA-MRSA/J has been detected in Tokyo, Niigata, western Japan, and Hong Kong. Our present study revealed the presence of this MRSA clone in Hokkaido, suggesting its nationwide spread.

The present study indicated the changing epidemiology of MRSA in northern Japan, revealing an increase of ST5/ST764 MRSA harboring ACME II′, emergence of MRSA with novel ACME II′-SCCmec CI and also ST8 CA-MRSA/J. Continuous surveillance to monitor their prevalence is necessary to be alert to these emerging MRSA clones.