Dissemination of methicillin-resistant staphylococci among healthy Japanese children

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA), regarded as a tenacious pathogen in the hospital, has recently become increasingly prevalent as a community pathogen. We evaluated the prevalence and characteristics of methicillin-resistant staphylococci in the Japanese community by testing nasal samples of 818 children of five day care centers and two kindergartens in three districts. We found that methicillin-resistant staphylococci are already prevalent among healthy children. Among 818 children, 35 children (4.3%) carried MRSA and 231 children (28.2%) carried methicillin-resistant coagulase-negative staphylococci (MRC-NS). The types of staphylococcal cassette chromosome mec (SCCmec) found among 44 MRSA isolates were as follows: type IIa, 11 isolates; type IIb, 19 isolates; and type IV, 14 isolates. The type IIb SCCmec element was a new SCCmec element found in this study. Eleven (25%) strains which belonged to clonal complex 5 (CC5) carried type IIa SCCmec, and they produced type 2 coagulase and toxic shock syndrome toxin 1. They were indistinguishable from health care-associated MRSA (H-MRSA) strains in Japan, represented by strain N315. On the other hand, 33 (75%) strains, most of which belonged to CC78 or CC91, carried small SCCmec elements, such as type IIb or type IV, and they produced type 1 or type 3 coagulase and exfoliative toxin. The data indicated that MRSA clones distinct from H-MRSA have disseminated in healthy children. The fact that MRC-NS strains were prevalent in the community suggested that they might serve as a reservoir for the SCCmec element carried by MRSA strains disseminated in the community.

FIG. 1.

Structural comparison of type IIa and type IIb SCCmec elements. The structures of the type IIa and type IIb SCCmec elements are illustrated based on the nucleotide sequences deposited in the DDBJ/EMBL/GenBank databases under accession nos. D86934 and AB127982, respectively. The entire SCCmec regions of the type IIb SCCmec elements were amplified by PCR with four sets of primers, indicated with arrowheads. The four sets of primers are as follows: cR1 and mA3, mA2 and ermA1 (5′-TGAAACAATTTGTAACTATTGA-3′), ermA3 (5′-TGGGTAAACCGTGAATATCGTGT-3′) and 2AJ1 (5′-ATTAGCCGATTTGGTAATTGAA-3′), and 2AJ2 (5′-TCGTACTTTGACGTAAATAGCCT-3′) and cD1 (5′-TAGTAAAGACTGTGAAATCTCATAT-3′). The nucleotide sequences of primers cR1, mA2, and mA3 were reported previously (25, 29). Type II SCCmec is defined as an SCCmec element which typically possesses a class A mec gene complex combined with a type 2 ccr gene complex. The element was further subtyped according to the differences in the nucleotide sequences of the junkyard 1 regions. The novel subtype of type II SCCmec element, type IIb, carried Tn554, which encodes macrolide-lincomycin-spectinomycin resistance, but did not carry pUB110, which encodes aminoglycoside resistance. Although IS256 was inserted upstream of the mecI gene, we are not sure whether or not it is happenstance that it was inserted in the element. The locations of the two sets of primers used for the identification of the type IIa SCCmec-specific J1 region and the type IIb SCCmec-specific J1 region are shown in bars. Two primers, primers 2a1 and 2a2, were constructed on the basis of the sequence of the kdp operon; and two primers, primers 2b1and 2b2, were constructed on the basis of the sequence of open reading frame S02.

FIG. 2.

Dendrogram of PFGE banding patterns of 44 MRSA isolates. A total of 44 MRSA strains were classified into eight pulsotypes. Pulsotypes of a representative strain of each type were compared by using the BioNumerics software program. The institutions and classes of children from whom MRSA strains were isolated are also indicated. The institution (kindergarten or day care center) to which a child belonged is indicated by the following abbreviations: M1, a child in Miyagi tested the first time; M2, a child in Miyagi tested the second time; S, a child in Saga; K, a child in Kyoto. Seven institutions have been subdivided according to the classes that they have. Their classes are indicated in parentheses by abbreviations. The names of the institutions are shown with letters, and the classes are indicated with numerals. Four institutions in Miyagi had multiple classes. They are indicated as a (a1 to a7), b (b1 to b7), c (c1 to c6), and d (d1 to d4). Two institutions in Saga and an institution in Kyoto are also indicated as a (a1 to a7) and b (b1 to b6) and a (a1 to a8), respectively.