In this study, we used three isolates from three epidemiologically unrelated patients. The first patient was a newborn male of low birth weight who became infected 20 h after birth. S. agalactiae was the predominant organism in nasal discharge, throat swab, and eye discharge. This strain was given reference number 02Z95 (=GTC1966).
The second patient was a 77-year-old male hospitalized due to an acute aortic dissection. In the hospital, he had been receiving intravenous hyperalimentation. After 1 month in the hospital, he was prescribed antibiotics (vancomycin and arbekacin) to cure a methicillin-resistant Staphylococcus aureus infection around the intravenous catheter. S. agalactiae was obtained from a throat swab after 2 months in the hospital. The number of this isolate was 02Z106 (=GTC1967).
The third patient was a 75-year-old female, admitted to the hospital for insertion of tension-free vaginal tape. On admission, S. agalactiae was detected in her urine. The strain was designated 02Z119 (=GTC2001).
We surveyed all medical records for these three patients to determine whether they had received any quinolones; however, we could not find any information.
The susceptibility testing of these isolates were carried out at the clinical laboratory section of the hospital. The MicroScan Walk Away system (Dade Behring Co., Tokyo, Japan) was used to determine the MICs of 33 antimicrobial agents, including 3 penicillins, 12 cephems, 3 aminoglycosides, 3 macrolides, 3 tetracyclines, 1 quinolone (levofloxacin [LVX]), and 1 glycopeptide (vancomycin). The three S. agalactiae isolates were susceptible to all antimicrobial agents used in this study except LVX (MIC > 8 μg/ml). To confirm the resistance to the quinolones, an E-test (AB Biodisk Sweden) was carried out for six classes of fluoroquinolones: LVX, ciprofloxacin, norfloxacin, ofloxacin, fleroxacin, and sparfloxacin. The MICs for the type strain (GTC1234T [=NCTC 8181T]) and three reference strains of S. agalactiae (GIFU10482, GIFU10483, and GIFU10484) were determined (Table ). However, three clinical isolates (GTC1966, GTC967, and GTC2001) were not inhibited in these tests, confirming that these isolates were multiply highly quinolone-resistant S. agalactiae strains.
MICs (E-test) of quinolones for S. agalactiae and mutations in gyrA and parC
To determine the QRDRs of gyrA
, we amplified DNA fragments from chromosomal DNA by PCR using previously reported PCR primers for gyrA
). However, the primers amplified the target fragments from S. pyogenes
but not from S. agalactiae
strains (GTC1234, GIFU10482, GIFU10483, and GIFU10484). Therefore, we designed new PCR primers from the region common to S. pyogenes
and S. agalactiae
: GyrA-forward, 5′ GACAAGTGAAATGAAAACGAG (positions 33 to 53); GyrA-reverse, 5′ CGCTCCATTGACTAATAAATTAGG (positions 484 to 507); ParC-forward, 5′ CAAAACATGTCCCTTGAGGA (positions13 to 32); and ParC-reverse, 5′ CTAGCTTTGGGATGATCAATCAT (positions 577 to 599). After confirmation of a single 474- or 586-bp amplification product of gyrA
, respectively, on 1% agarose gels, sequences were determined with an automatic sequencer (model 3100; Applied Biosystems) with a dye terminator reaction kit (Applied Biosystems). DNA and protein sequence comparisons were done with DNASIS software (Hitachi Software Co., Yokohama, Japan).
First, we estimated the specificity of our PCR primers using the type strains of several beta-hemolytic streptococci. Subsequently, our primers for both gyrA
could amplify the target fragments from the following species: S. agalactiae
]), Streptococcus pyogenes
]), Streptococcus equi
]), Streptococcus equi
]), Streptococcus iniae
]), Streptococcus canis
]), Streptococcus dysgalactiae
]), S. dysgalactiae
]), Streptococcus porcinus
]), and Streptococcus difficile
]). All of these species belong to the pyogenic group of the genus Streptococcus
). The species of the anginosus group are also beta-hemolytic, although our primers did not amplify fragments from the type strain of each species in this group (S. anginosus
]; Streptococcus intermedius
]; and Streptococcus constellatus
]). Because the anginosus group is phylogenetically distant from the pyogenic group (7
), the sequences of gyrA
may be slightly different from those in S. agalactiae
and S. pyogenes
Comparative amino acid sequences deduced from gyrA and parC (including the QRDR) from representative strains of both quinolone-susceptible and -resistant S. agalactiae strains and some other beta-hemolytic streptococci are shown in Fig. and , respectively.
FIG. 1. Comparison of the deduced amino acid sequences of a region of gyrA containing a QRDR from quinolone-susceptible and -resistant strains of S. agalactiae and other beta-hemolytic streptococci. SAGAL, S. agalactiae; SDIFF, S. difficile; SPYOG, S. pyogenes (more ...)
Amino acid sequences deduced from a region of parC containing a QRDR from quinolone-susceptible and -resistant strains of S. agalactiae and other beta-hemolytic streptococci. Symbols and abbreviations are as in Fig. .
All four quinolone-susceptible S. agalactiae
strains (GTC1234, GIFU10482, GIFU10483, and GIFU10484) shared the same deduced amino acid sequences for the QRDRs of both gyrA
. Similarly, three isolates of highly quinolone-resistant strains (GTC1966, GTC1967, and GTC2001) had identical amino acid sequences. However, compared with susceptible strains, these quinolone-resistant S. agalactiae
strains carried double point mutations of DNA with the following inferred amino acid substitutions involving the QRDRs of gyrA
; Ser-81 to Leu (TCA → TTA) for gyrA
and Ser-79 to Phe (codon TCC → TTC) for parC
. The mutations at these positions were previously described as contributing to quinolone resistance (4
Three major mutation sites have been previously reported for quinolone-resistant streptococci, namely, position 81 in gyrA
and positions 79 and 83 in parC
. Yokota et al. found that some quinolone-resistant S. pneumoniae
strains had other mutations, including Ser-114 to Gly in gyrA
and Ser-52 to Gly, Asn-91 to Asp, and Glu-135 to Asp in parC
). We concluded that these mutations were not related to quinolone resistance, because, as shown in Fig. and , many quinolone-susceptible streptococci had the same amino acid sequences.
Surprisingly, S. agalactiae
exhibited sequences different from those of other beta-hemolytic streptococci: S. agalactiae
strains had Met in position 132 of the gyrA
product, whereas all other beta-hemolytic streptococci in this study had Leu (Fig. ). All beta-hemolytic streptococci and even S. pneumoniae
strains have same amino acid at positions 69, 97, 99, and 113 of the parC
product (Ile, Ile, Val, and Pro, respectively); however, S. agalactiae
strains (seven strains including the type strain) and the type strain of S. difficile
have different amino acids (Val, Thr, Ile, and Ala, respectively, at these positions) (Fig. ). We cannot easily explain why only S. agalactiae
and S. difficile
have different amino acids at these positions. According to a FASTA homology search (12
) on the DDBJ website (http://www.ddbj.nig.ac.jp
), there was no more closely related organism than beta-hemolytic streptococci (data not shown). At this time, we do not expect that the genes were transferred from other organisms.
We were also surprised that S. agalactiae
and S. difficile
shared identical amino acid sequences of QRDRs in both gyrA
(Fig. and ). There were numerous silent nucleotide base substitutions, especially in parC
: for example, only two amino acid differences were found between S. equi
and S. equi
, although there were 21 silent base substitutions (data not shown). In the case of S. agalactiae
and S. difficile
, only one base difference was found. Strains of S. agalactiae
have been isolated from homeothermic animals, including humans, whereas S. difficile
has been isolated from poikilothermic animals, such as fish (2
). Horizontal gene transfer is one possible explanation for the presence of the same sequences in these two species.
Munoz and Campa had reported that parC
was the primary target of quinolones in S. pneumoniae
). In this study we isolated three resistant strains of S. agalactiae
, with the same point mutation in both gyrA
. We cannot tell which gene is the primary target of the quinolones in S. agalactiae
on the basis of our isolates; further study is needed.