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Antimicrobial Agents and Chemotherapy, June 2008, p. 2061-2068, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.01150-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Methicillin-Resistant Staphylococcus saprophyticus Isolates Carrying Staphylococcal Cassette Chromosome mec Have Emerged in Urogenital Tract Infections

Masato Higashide,^1,2 Makoto Kuroda,^1,3* Carlos Takashi Neves Omura,¹ Miyuki Kumano,¹ Saburo Ohkawa,⁴ Sadahiro Ichimura,⁵ and Toshiko Ohta¹

Department of Microbiology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan,¹ Kotobiken Medical Laboratories Inc., 445-1 Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan,² Laboratory of Bacterial Genomics, Center for Pathogen Genomics, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan,³ Department of Microbiology, Kohjin Bio Co., Ltd., 5-1-3 Chiyoda, Sakado, Saitama 350-0214, Japan,⁴ Department of Bacteriology, BML, Inc., 1361-1 Matoba, Kawagoe-shi, Saitama 350-1101, Japan⁵

Received 30 August 2007/ Returned for modification 28 October 2007/ Accepted 15 March 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Staphylococcus saprophyticus is a uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in female outpatients. We investigated the dissemination and antimicrobial susceptibilities of 101 S. saprophyticus isolates from the genitourinary tracts of patients in Japan. Eight of these isolates were mecA positive and showed β-lactam resistance. Pulsed-field gel electrophoresis showed that only some isolates were isogenic, indicating that the mecA gene was apparently acquired independently by mecA-positive isolates through staphylococcal cassette chromosome mec (SCCmec). Type determination of SCCmec by multiplex PCR showed a nontypeable element in the eight mecA-positive isolates. Sequence analysis of the entire SCCmec element from a prototype S. saprophyticus strain revealed that it was nontypeable with the current SCCmec classification due to the novel composition of the class A mec gene complex (IS431-mecA-mecR1-mecI genes) and the ccrA1/ccrB3 gene complex. Intriguingly, the attachment sites of SCCmec are similar to those of type I SCCmec in S. aureus NCTC 10442. Furthermore, the genes around the mec gene complex are similar to those of type II/III SCCmec in S. aureus, while those around the ccr gene complex are similar to those of SCC15305RM found in S. saprophyticus ATCC 15305. In comparison with known SCCmec elements, this S. saprophyticus SCCmec is a novel type.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Staphylococcus saprophyticus is a member of the coagulase-negative staphylococci (CoNS), which frequently cause uncomplicated urinary tract infections (UTIs) in young and middle-aged female outpatients (8, 12, 15, 18, 21, 22, 23). Unlike most other CoNS, S. saprophyticus is rarely resistant to most antibiotics active against gram-positive organisms (10, 17).

Although the UTIs caused by S. saprophyticus have been well documented, the antimicrobial resistance and dissemination of this species are not well studied. The study described here investigated the current dissemination and antimicrobial resistance of S. saprophyticus isolates recovered from the urogenital tracts of Japanese patients. In addition, the characterization of a new type of staphylococcal cassette chromosome mec (SCCmec) element from a mecA-positive S. saprophyticus was carried out.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial isolates. From April to December 2003, a total of 101 S. saprophyticus isolates were recovered from urine specimens (94 isolates) of patients with acute cystitis with bacterial counts of 10⁴ CFU/ml or vaginal specimens (7 isolates) of patients with from bacterial vaginosis- or candidiasis-related symptoms at the clinical microbiology laboratories of 65 different Japanese hospitals. All isolates were from different patients, and duplicate isolates from the same patient were excluded. In addition, specimens also yielding gram-negative bacteria, which are often isolated from patients with either uncomplicated or complicated UTIs, were not considered for further isolation of CoNS, including S. saprophyticus. These isolates were identified as CoNS by means of multiple assays, including Gram staining, catalase production, coagulase production (Eiken, Japan), DNase production (Eiken), growth on egg yolk mannitol salt agar (Becton Dickinson), and the novobiocin susceptibility test (Showa, Japan). The final identification was carried out with the API ID32 Staph system in a mini API system (bioMérieux, France).

Antimicrobial susceptibility tests. The MICs were determined by the agar dilution method, as described in CLSI document M7-A7 (3). The testing range of the antimicrobial concentrations was from 0.06 to 256 µg/ml. Ampicillin, cefoxitin, kanamycin, ofloxacin, and oxacillin were purchased from Sigma (St. Louis, MO). Cefazolin, erythromycin, and vancomycin were purchased from Wako (Japan). The following antibiotics were obtained from their respective manufacturers: arbekacin (Meiji, Japan), clarithromycin (Taisho, Japan), fosfomycin (Meiji, Japan), imipenem (Banyu, Japan), teicoplanin (Fujisawa, Japan), and trimethoprim-sulfamethoxazole (Shionogi, Japan).

In the case of oxacillin, Mueller-Hinton agar was supplemented with 2% (wt/vol) sodium chloride. The MIC of fosfomycin was determined on Mueller-Hinton agar supplemented with 25 µg/ml of glucose-6-phosphate. The agar plate was incubated at 35°C for 24 h.

Detection of mecA-positive S. saprophyticus isolates. The mecA-positive isolates were detected by dot blot hybridization, according to a previously described method (14). Briefly, S. saprophyticus cell lysates were denatured with alkaline buffer and were then spotted onto a GeneScreen Plus hybridization membrane (NEN). The membrane was subjected to hybridization with a mecA gene-specific probe labeled by use of the AlkPhos direct labeling kit (GE Healthcare, Buckinghamshire, England), followed by detection with CDP-Star. The mecA PCR product and methicillin-resistant Staphylococcus aureus (MRSA) N315 cell lysate were used as a positive control.

PFGE analysis. A pulsed-field gel electrophoresis (PFGE) plug mold was prepared by using a GenePath Group 1 reagent kit for CoNS (Bio-Rad), according to the manufacturer's instruction. The chromosomal DNA in the plug mold was digested with the SmaI restriction enzyme. PFGE was performed under the following conditions: 6 V/cm, ±60° angle, 5.3-s initial time, 34.9-s final time, and a run time of 20 h at 14°C with 0.5x Tris-borate-EDTA buffer (CHEF DRIII apparatus; Bio-Rad).

A dendrogram showing the similarity of the PFGE profiles was obtained by the unweighted pair group method with the arithmetic mean with GelCompar software (Applied Maths, Kortrijk, Belgium).

The mecA-positive SmaI-digested DNA fragment was detected by Southern hybridization method with a mecA gene-specific probe labeled by use of a AlkPhos direct labeling kit (GE Healthcare).

Multiplex PCR for type assignment of the SCCmec element and mec and ccr gene complexes. The multiplex PCR for the type assignment of SCCmec was performed as described in the report of Oliveira and de Lencastre (19), but the thermostable DNA polymerase was exchanged with Phusion high-fidelity DNA polymerase (Finzyme, Espoo, Finland). Multiplex PCR for type assignment of the mec or the ccr gene complex was performed by the procedure of Kondo et al. (11).

Sequencing of SCCmec element in S. saprophyticus TSU33. The SCCmec element from the TSU33 strain of S. saprophyticus was partially amplified by PCR with ExTaq DNA polymerase (Takara, Japan) and oligonucleotide primers (Table 1) corresponding to the consensus nucleotide sequences of the orfX, mecA, and ccrB genes among the SCCmec elements. The PCR products for DNA sequencing were obtained as follows. The amplicon between orfX and mecA was amplified with primers orfX-201F and mecA-33F. The amplicon between mecA and ccrB was amplified with primers mecA-381R and ccrB-F. To obtain an adjacent DNA fragment downstream of the ccr locus, inverse PCR was performed with HindIII-digested/self-ligated chromosome DNA from strain TSU33 and primers ccr-InvF and mecAccr-R4-2 under the following reaction conditions: predenaturation at 94°C for 30 s; 30 cycles at 94°C for 30 s, 55°C for 1 min, and 68°C for 12 min; postextension at 72°C for 4 min; and soaking at 4°C. To obtain the adjacent downstream DNA region, further inverse PCR walking was performed by using the same PCR parameters described above and with the following primers and template DNA: primers 2nd-InvF and 2nd-InvR with EcoRI-digested/self-ligated chromosomal DNA as the template and primers 3rd-InvF and 3rd-InvR with PstI-digested/self-ligated chromosome DNA as the template. The DNA sequences on both strands were determined by the primer walking method with the BigDye Terminator cycle sequencing kit (version 1.1) on an ABI 310 DNA sequencer (Applied Biosystems).

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TABLE 1. Oligonucleotide primers used in this study

Bioinformatic analysis of SCCmec elements. Putative open reading frames (ORFs) were predicted by use of the GLIMMER (version 2.0) program (5), with manual determination of potential ribosome binding sequences. Functional assignments of the predicted ORFs were based on a search for homology against the sequences in the nonredundant protein database by use of BLASTp program (1). Pairwise alignment of the SCCmec elements was performed by a search for homology (1) between the elements by use of the BLASTn program, followed by visualization of the aligned images with the ACT program (2). The phylogenetic tree of the amino acid sequences of the CcrA and CcrB recombinases was obtained by analysis with the ClustalX program with bootstraps of 1,000 (20).

Nucleotide sequence accession number. The complete sequence and annotation of the SCCmec element of S. saprophyticus TSU33 have been deposited in the DDBJ database (accession number AB353724).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Isolation and identification of S. saprophyticus from urine and vaginal specimens. S. saprophyticus is a novobiocin-resistant CoNS (NRCoNS) strain. Overall, 96 NRCoNS isolates were obtained from 6,743 urine specimens of patients with acute cystitis with bacterial counts of 10⁴ CFU/ml. The vaginal flora could be one of the potential reservoirs for S. saprophyticus UTIs; isolates of nine species of NRCoNS were isolated from 12,153 vaginal specimens. Of the 105 NRCoNS isolates recovered, 101 were identified as S. saprophyticus, 3 were identified as S. cohnii, and 1 was identified as S. sciuri. Other NRCoNS species, such as S. ariettae, S. equorum, S. gallinarum, S. kloosii, and S. xylosus, were not detected in this study.

Antimicrobial susceptibilities of the S. saprophyticus isolates. We investigated the antimicrobial susceptibilities of the 101 S. saprophyticus isolates (Table 2). All isolates showed oxacillin MICs in the resistance range (0.5 µg/ml), in accordance with CLSI document M100-S18 guidelines for CoNS (4). However, the oxacillin susceptibility pattern exhibited a clear bimodal distribution, as we reported previously (7). Screening for the presence of the mecA gene by dot blot hybridization and PCR showed that the 93 isolates with oxacillin MICs in the range of 0.5 to 4 mg/liter were all mecA negative, while the 8 isolates with oxacillin MICs greater than 64 mg/liter were mecA positive (data not shown). These mecA-positive isolates showed relatively high MICs with respect to β-lactams, macrolides, and fosfomycin but not to the other antibiotics tested (Table 3).

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TABLE 2. Distribution of MICs of antibiotics tested for 101 Staphylococcus saprophyticus isolates

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TABLE 3. Characteristics of eight mecA-positive S. saprophyticus isolates

One mecA-positive isolate was from a vaginal specimen, and the other seven mecA-positive isolates were from urine specimens. Two of the eight isolates were from urine specimens from males, and 1 of them was from an inpatient. No mecA-negative isolate was found in males, suggesting that the UTIs caused by S. saprophyticus in males could be associated with hospitalization, as reported previously (18).

Intriguingly, in comparison with other staphylococci, fosfomycin resistance was observed in all S. saprophyticus isolates (Table 2), suggesting that resistance to this drug is an intrinsic phenotype, as reported previously (10).

Distribution of multiple clones of mecA-positive S. saprophyticus. The PFGE analysis showed different SmaI digestion profiles for the chromosomal DNAs of the mecA-positive S. saprophyticus isolates. Three of the eight isolates showed identical or similar PFGE profiles (Fig. 1), suggesting clonal relatedness. These isolates were from different patients and hospitals, suggesting the potential for clonal spread. Southern hybridization with the mecA gene-specific probe showed a uniform pattern for these three isolates (Fig. 1). On the other hand, the results of PFGE profiling and Southern hybridization for the other five isolates revealed different patterns, suggesting that multiple mecA-positive S. saprophyticus strains might circulate in Japanese communities. Similar to our observation, Widerstrom et al. also reported that multiple clones of S. saprophyticus were associated with lower UTIs in women (24).

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FIG. 1. PFGE analysis of eight mecA-positive S. saprophyticus strains. S. saprophyticus ATCC 15305 and S. aureus N315 were also included in this analysis as reference strains. Composite clustering of the PFGE profiles is shown along with the dendrogram with the percentage of tree branch reliability obtained by the unweighted pair group method with the arithmetic mean method with bootstrap analysis. Arrowheads indicate the positive band detected by Southern hybridization with the mecA-specific probe. S. saprophyticus type strain ATCC 15305 is mecA negative. The profiles of three isolates are enclosed by a broken line; these are probably isogenic strains.

Type assignment of the SCCmec element in S. saprophyticus. The SCCmec types of the eight mecA-positive isolates could not be assigned by the multiplex PCR approach described by Oliveira and de Lencastre (19) (Fig. 2A), because these yielded a combination of DCS and mecI amplifcation products that was not consistent with those of known types of SCCmec elements. Further classification of a mec and a ccr gene complex by multiplex PCR methods (11) confirmed that the eight isolates were nontypeable according to current schemes, because all eight isolates possessed a class A mec gene complex (Fig. 2B) but yielded no amplification product for known ccr genes (Fig. 2C).

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FIG. 2. Multiplex PCRs for the type assignment of the SCCmec element of mecA-positive S. saprophyticus (A) (19), for the determination of a mec class (B) (11), and for the determination of a ccr class (C) (11).

To characterize the novel SCCmec type of S. saprophyticus, we selected vaginal isolate TSU33, because the vagina might be a potential reservoir for UTIs; in addition, TSU33 could be the prototype of the clonally spreading strain among the three isogenic isolates (strains TSU18, TSU33, and TSU57) described above.

The sequence of SCCmec from TSU33 was 23,743 bp long and was composed of 26 ORFs (Table 4 and Fig. 3). Possible attachment sequences of attC (on the chromosome DNA) and attSCC (on the SCC element) for SCCmec integration were investigated by a search for direct repeats, which revealed that the attC/attSCC of this SCCmec element was most similar to that of type I SCCmec of S. aureus NCTC 10442 (Fig. 4) (9). In addition, pairwise alignment of the sequence around attC and attSCC showed that possible inverted repeat sequences (inverted repeats IR-L and IR-R), which are predicted to be the cis elements for CcrAB recombinases, were also similar to those of type I SCCmec of S. aureus NCTC 10442 rather than to those of the other types (Fig. 4). SCCmec in TSU33 carries the class A mec gene complex (IS431-mecA-mecR1-mecI genes) that is found in type II SCCmec of S. aureus N315 or type III SCCmec of S. aureus 85/2082 (Table 4 and Fig. 3) (9). Around the mec gene complex, orf2, located in junkyard region J3 between orfX and the mec gene complex, was very similar to that of type I SCCmec (S. aureus NCTC 10442) and type II SCCmec (S. aureus N315) (Table 4 and Fig. 3), while it did not show any similarity to SCC_15305RM in S. saprophyticus ATCC 15305 (Fig. 3) (13).

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TABLE 4. Similarities of ORFs in the SCCmec element and in the region adjacent to the chromosome in S. saprophyticus TSU33

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FIG. 3. Schematic representation of the SCCmec elements and pairwise alignment of their nucleotide sequences. The arrows on the upper, middle, and lower lines show the organization of ORFs in the SCC15305RM of S. saprophyticus ATCC 15305, which is a mecA-negative clinical isolate; SCCmec in S. saprophyticus TSU33; and type I SCCmec of S. aureus NCTC 10442, respectively. Pairwise alignment was analyzed by the use of ACT software and the results from a search for homology between nucleotide sequences of the SCC elements by use of the BLASTn program. Matched sequences are shown as pink bars (same orientation) and dark blue bars (inverted orientation), and those with a BLASTn score of less than 200 were excluded.

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FIG. 4. Pairwise alignment of the attachment site of SCCmec elements. Possible attachment sequences of attC (on the chromosomal DNA) and attSCC (on the SCC element) for SCCmec integration are boxed. As an alignment control, S. aureus NCTC 8325 was used as the SCCmec-negative strain. The same nucleotide sequence compared with that of SCCmec in strain TSU33 is shown in boldface. Possible inverted repeat sequences (inverted repeats IR-L and IR-R) adjacent to attC and attSCC, which are predicted to be the cis elements for CcrAB recombinases, are shown as arrows under the nucleotide sequences. The nucleotide sequences of the SCCmec elements and the genome sequence have the following DDBJ database accession numbers: NCTC 10442, AB033763; N315, D86934; 85/2082, AB037671; WIS, AB121219; and NCTC 8325, NC_007795.

The ccr genes and their compositions are some of the key features used for categorizing SCCmec types. CcrA and CcrB of SCCmec in strain TSU33 could be categorized as CcrA1 and CcrB3, respectively, and had amino acid similarities (Fig. 5) but were not perfectly identical to the known subclasses of CcrA and CcrB recombinases (Table 4). Apart from the amino acid similarity, this combination of ccr genes, such as ccrA1 and ccrB3, has not yet been reported. In fact, nontypeable ccr gene complexes seem to be widely distributed among CoNS (6). The ORFs around the ccr genes showed a high degree of similarity to SCC_15305RM in S. saprophyticus ATCC 15305 but were different from those found in other SCCmec elements, including type I SCCmec, as shown in Fig. 3.

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FIG. 5. Phylogenetic tree obtained by the alignment of amino acid sequences among Ccr recombinases. Basically, Ccr recombinases are classified as CcrA, CcrB, and CcrC and are categorized into subtypes, for example, CcrA1, as shown next to the circles. The Staphylococcus strains possessing Ccr recombinases are as follows: S. saprophyticus ATCC 15305 and TSU33; S. aureus NCTC 10442, N315, methicillin-susceptible strain 476, methicillin-resistant strain 252, 85/2082, MW2, WIS, and HDE288; S. epidermidis ATCC 12228; and S. hominis ATCC 27844. The scale is 0.1 amino acid replacements per site.

The ORFs of the junkyard J2 region between mecI and ccrB showed some similarity to those of type III SCCmec in S. aureus 85/2082, while the ORFs of the junkyard J1 region between ccrA and orf28, adjacent to the attC site, did not show significant similarity to those present in other SCCmec types (Table 4).

Concluding remarks. Regarding the antimicrobial susceptibility of S. saprophyticus, we previously reported on the prevalence of β-lactam-resistant S. saprophyticus isolates in Japan (7). In fact, there are few reports on mecA-mediated resistance in S. saprophyticus. One of the reasons could be that S. saprophyticus is not a commensal bacterium that colonizes human skin; thus, it is less likely to have become antimicrobial resistant by horizontal gene transfer from MRSA or methicillin-resistant S. epidermidis. Indeed, the SCCmec element of TSU33 shows a unique gene organization in the ccr gene complex. In addition, recent reports (6, 16) and our results suggest that CoNS are more likely to contain several representatives of different ccr complexes.

 
This work was supported by grant from Kotobiken Medical Laboratories Inc.

 
* Corresponding author: Mailing address: Laboratory of Bacterial Genomics, Center for Pathogen Genomics, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: 81-3-5285-1111. Fax: 81-3-5285-1166. E-mail: makokuro{at}nih.go.jp

Published ahead of print on 24 March 2008.

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Antimicrobial Agents and Chemotherapy, June 2008, p. 2061-2068, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.01150-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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