Distribution of Staphylococcal Cassette Chromosome (SCC) mec Element Types in Fusidic Acid-Resistant Staphylococcus epidermidis and Identification of a Novel SCC₇₆₈₄ Element

Abstract

We analyzed the staphylococcal cassette chromosome mec (SCCmec) types of 143 fusidic acid- and methicillin-resistant Staphylococcus epidermidis isolates. The most frequent SCCmec type was SCCmec III/SCCHg (53%), followed by SCCmec IV (29%). Clonal spreading of SCCmec III/SCCHg strains contributed to the increased prevalence of SCCmec III. A novel non-mec SCC structure, SCC₇₆₈₄, adjacent to SCCmec III, which carries a new ccrC allotype (ccrC3 allele 1) and contains heavy metal resistance genes, was identified in 14 isolates.

TEXT

Methicillin resistance in staphylococci results from the production of an alternative penicillin-binding protein 2a (PBP 2a) with low affinity for β-lactam antibiotics, encoded by the mecA gene. The mecA gene is located on a mobile genetic element known as the staphylococcal cassette chromosome mec (SCCmec) (1). It has been suggested that methicillin-susceptible Staphylococcus aureus acquires SCCmec from methicillin-resistant coagulase-negative staphylococci (CoNS) and becomes methicillin-resistant S. aureus (MRSA) (2, 3). The structures and types of SCCmec in CoNS are usually more complex than in S. aureus. In addition to SCCmec, non-mec SCCs composed of ccr genes and resistance genes other than mecA have been reported, such as SCCHg, SCCfusC (4), and SCCpbp4 (5).

We previously found that the majority of fusidic acid-resistant Staphylococcus epidermidis isolates were also resistant to methicillin (6). To understand the distribution of SCCmec types, a total of 155 fusidic acid-resistant S. epidermidis isolates, including 141 clinical isolates that were collected between 2008 and 2010 in the Bacteriology Laboratory of the National Taiwan University Hospital (6) and 14 commensal isolates (7), were tested. The mecA gene was detected in 143 isolates, including 137 clinical isolates and 6 commensal isolates. Among 137 clinical isolates, 132 carried fusB, 4 carried fusC, and 1 had an fusA point mutation, all of which have been studied and published (6). All of the 6 commensal methicillin-resistant isolates carried fusB.

The SCCmec types of 143 mecA-positive isolates were determined by standard methods (1, 8–12), and the results are listed in Table 1. The most frequent SCCmec type was SCCmec III/SCCHg (n = 76, 53%), followed by SCCmec IV (n = 41, 29%). This result is different from those of other reports in which SCCmec IV was usually dominant in methicillin-resistant S. epidermidis (MRSE) (13, 14). However, the isolates tested in the present study were all fusidic acid-resistant rather than general MRSE. There were 17 SCCmec III isolates that lacked SCCHg. Of them, 3 carried only SCCmec III, and 14 contained an additional novel structure, SCC₇₆₈₄ (described later). All of the 6 mecA-positive commensal isolates were SCCmec type IV. An additional ccr gene was found in 2 SCCmec type IV/ccrA1B1, 1 SCCmec type IV/ccrC, and 2 SCCmec type IV/SCCfusC isolates. One SCCmec type V_T/SCCfusC was identified. It is not uncommon for mecA-positive CoNS to carry multiple ccr copies or no ccr genes (3, 15, 16). SCCfusC adjacent to SCCmec III was previously found in MRSA (4). In the present study, SCCfusC was linked to SCCmec IV or SCCmec V_T in S. epidermidis, which has not been reported before, suggesting that insertions of SCCmec and SCCfusC were independent. In 4 SCCmec nontypeable isolates, no ccr gene was detected. For SCCmec nontypeable isolates, there are two possibilities: the isolates may have a novel type or the target sites for the primers may have been altered. In a study in Norway, ccr-nontypeable S. epidermidis isolates were reported 52% of the time (3).

TABLE 1.

SCCmec types and pulsotypes among 143 fusidic acid-resistant S. epidermidis isolates

SCCmec type	Other SCC, additional ccr gene, or mec complex	No. of isolates	PFGE pattern(s) (no. of isolates)
III	SCCHg	76	A (21), B (6), D (36), E (1), F (8), G (2), AC (1), AM (1)
III		3	A (2), D (1)
III	SCC7684	14	I (11), J (1), N (1), P (1)
IV		36	A (1), H (1), K (1), Q (3), T (2),b V (2), W (1), X (1),Y (2), Z (2), AA (4), AB (1),b AD (2), AE (2), AF (3), AG (3), AH (1), AI (1), AQ (3)b
	ccrA1B1	2	Q (1), R (1)
	ccrC	1	L (1)
	SCCfusC	2	X (1), Y (1)
IV varianta		3	L (1), M (1), AA (1)
VT	SCCfusC	1	X (1)
VIII		1	A (1)
NT1	mec complex A	3	I (1), N (1), U (1)
NT2	mec complex B	1	O (1)
Total		143

^aThis type is the large size of mec complex B.

^bThese are commensal isolates collected from healthy volunteers.

Pulsed-field gel electrophoresis (PFGE) analysis divided 155 S. epidermidis isolates (143 MRSE and 12 methicillin-susceptible S. epidermidis [MSSE] isolates) into 43 clusters (Table 1). Pulsotype D was the most frequent (37/155, 24%), followed by pulsotype A (25/155, 16%). Most of the isolates that carried SCCmec III/SCCHg belonged to pulsotypes A (21/76, 28%) and D (36/76, 47%). Clonal spreading of SCCmec III/SCCHg strains may contribute to the increased prevalence of SCCmec III. Eleven of 14 isolates that contained SCCmec III/SCC₇₆₈₄ were pulsotype I. Isolates of SCCmec IV were distributed among different pulsotypes. Six commensal isolates that carried SCCmec IV were distributed in the following three pulsotypes: T, AB, and AQ. Some isolates with different SCCmec types clustered in the same pulsotype, such as pulsotype A (SCCmec III/SCCHg, 21 isolates; SCCmec III, 2 isolates; SCCmec IV, 1 isolate; SCCmec VIII, 1 isolate), indicating the possibility of the intraspecies transfer of SCCmec.

A total of 17 isolates were SCCmec type III but lacked SCCHg, which was the third most frequent type after SCCmec III/SCCHg and SCCmec IV. One isolate, NTUH-7684, was chosen for whole-genome sequencing to determine the possible novel composite SCCmec III-related element. A 56,238-bp element was found abutting the SCCmec III in NTUH-7684 (Fig. 1). The sequence of SCCmec III in NTUH-7684 showed 98.94% identity with that in S. aureus 85/2082, except for the truncated J3 region and the intact mecR1 and mecI, which were truncated in S. aureus 85/2082. A novel allotype of ccrC was identified. The 1,686-bp ccrC sequence was closest to that of the ccrC1 allele 10 in S. aureus TW20 (GenBank accession no. GQ902038; 69.69% identity) (Fig. 2) and showed 69.45% identity to ccrC1 allele 2 in S. aureus TSGH17 (GenBank accession no. AY894416). The ccrC gene in NTUH-7684 is designated ccrC3 allele 1 according to the 85% cutoff value. The phylogenetic tree based on concatenated sequences of ccrC genes indicated that ccrC3 allele 1 was phylogenetically separated from ccrC1 and ccrC2 (Fig. 2). The ccrC3 allele 1 was detected in 14 of 17 isolates with SCCmec type III lacking SCCHg by PCR using a pair of primers (SE-ccrC3-359F, 5′-GCGAAATGATGATAGGAGCA-3′, and SE-ccrC3-1368R, 5′-ATTCATAGCCTCAGCCTGC-3′). Isolates (11/14, 79%) that carried SCCmec III/SCC₇₆₈₄ mostly belonged to pulsotype I, indicating clonal spreading. The presence of the ccrC3 allele 1 indicated that the element was a non-mec SCC, which was referred to as SCC₇₆₈₄.

FIG 1.

Genetic organization of SCCmec III/SCC₇₆₈₄ in S. epidermidis NTUH-7684 (GenBank accession no. LC085180) compared with SCCmec III/SCCHg (GenBank accession no. AB037671) in S. aureus. Genes are shown according to their sequences. The predicted integration site sequences are indicated by arrows. Homologous regions between SCCmec are shown in shaded areas, and the numbers in the shaded areas show percent identities between the corresponding sequences.

FIG 2.

Phylogenetic relationships for the ccrC genes and sequence identity compared with ccrC3 allele 1 in S. epidermidis NTUH-7684. The phylogenetic tree was generated by using the neighbor-joining method with the MEGA6 package. Numbers at nodes are confidence levels expressed as percentages of occurrence in 500 bootstrapped resamplings. Scale bars indicate the evolutionary distance between sequences, determined by measuring the lengths of the horizontal lines connecting two organisms.

SCC₇₆₈₄ contained many heavy metal (cadmium, mercury, copper, and arsenic) resistance genes, genes associated with type I restriction modification systems (hsdR, hsdS, hsdM, IS431), and genes encoding hypothetical proteins. The region containing mercury resistance genes flanked by IS431 showed high similarity (93.12% identity) to the SCCHg region in S. aureus 85/2082. The 18-bp integration site sequences (ISSs), GAAGC(A/T/G)TA(T/C)CA(T/C)AA(A/G)T(A/G)A, were found at the end of SCCmec III and SCC₇₆₈₄ (Fig. 1). The SCCmec III and SCC₇₆₈₄ elements were flanked by 15-bp imperfectly matched direct repeats (DRs), (A/C)GAAGC(A/T/G)TA(T/C)CA(T/C)AA, which have also been found in other SCC elements (17, 18), suggesting that the two SCC elements integrated into the chromosome independently.

In conclusion, this is the first study to investigate the distribution of SCCmec types among fusidic acid-resistant S. epidermidis isolates. PFGE analyses of isolates carrying SCCmec III/SCCHg or SCCmec III/SCC₇₆₈₄ indicate that each showed clonal spreading. The SCC₇₆₈₄ element possessed many genes associated with heavy metal resistance, which may provide an advantage for bacterial survival. Our findings highlight the importance of characterizing the SCC-related elements in S. epidermidis.

Nucleotide sequence accession number.

The SCC₇₆₈₄ sequence from the S. epidermidis clinical isolate NTUH-7684 was deposited in GenBank under accession no. LC085180.