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Antimicrobial Agents and Chemotherapy, December 2006, p. 4077-4086, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00847-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Characteristics and In Vitro Susceptibility to Antimicrobial Agents, Including the Des-Fluoro(6) Quinolone DX-619, of Panton-Valentine Leucocidin-Positive Methicillin-Resistant Staphylococcus aureus Isolates from the Community and Hospitals{triangledown}

Tatsuo Yamamoto,1* Soshi Dohmae,1 Kohei Saito,1 Taketo Otsuka,1 Tomomi Takano,1 Megumi Chiba,2 Katsuko Fujikawa,2 and Mayumi Tanaka2

Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata,1 Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan2

Received 12 July 2006/ Returned for modification 17 August 2006/ Accepted 4 October 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Highly virulent, community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strains with Panton-Valentine leucocidin (PVL) genes have been found increasingly worldwide. Among a total of 2,101 MRSA strains isolated from patients in hospitals in Japan, two were positive for PVL genes. One strain was identified as a community-acquired MRSA strain with genotype sequence type 30 (ST30) and spa (staphylococcal protein A gene) type 19 from Japan and was resistant only to ß-lactam antimicrobial agents. The other strain was closely related to PVL+ multidrug-resistant, hospital-acquired MRSA strains (ST30, spa type 43) derived from nosocomial outbreaks in the 1980s to 1990s in Japan but with a divergent sequence type, ST765 (a single-locus variant of ST30). Twenty-two PVL+ MRSA strains, including those from Japan and those from other countries with various sequence types (ST1, ST8, ST30, ST59, and ST80) and genotypes, were examined for susceptibility to 31 antimicrobial agents. Among the agents, DX-619, a des-fluoro(6) quinolone, showed the greatest activity, followed by rifampin and sitafloxacin, a fluoroquinolone. The data suggest that DX-619 exhibits a superior activity against PVL+ MRSA strains with various virulence genetic traits from the community as well as from hospitals.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Methicillin-resistant Staphylococcus aureus (MRSA), alternatively called hospital-acquired MRSA (HA-MRSA), has been a major cause of nosocomial infections since 1961 (5). Besides this, since 1997 to 1999 (6), infection due to new highly virulent MRSA, called community-acquired MRSA (CA-MRSA), has become a major concern worldwide (52, 56). CA-MRSA infection is associated with skin and soft tissue infection in young, otherwise healthy people (33, 52, 56) and with more severe diseases, such as osteomyelitis or necrotizing pneumonia (preceded by influenza or influenza-like prodromes) (11, 16, 56).

CA-MRSA carries genes for Panton-Valentine leucocidin (PVL) (52, 56), which causes apoptosis and necrosis in human polymorphonuclear cells (19), and possesses limited types of methicillin resistance locus (staphylococcal cassette chromosome mec [SCCmec]), such as SCCmecIV or SCCmecV (10, 52, 56). There are geographically two types of CA-MRSA: one being rather continent specific (e.g., multilocus sequence type 1 [ST1] and ST8, found mostly in the United States, and ST80, found mostly in Europe [30, 52]) and the other worldwide (ST30, found in the United States, Europe, Oceania, Japan, and Brazil [12, 31, 41, 48, 52]). CA-MRSA is also isolated from hospitals (2, 37, 44).

In Japan, MRSA strains at the time of nosocomial outbreaks ("hospital MRSA panics") in the late 1980s and early 1990s were largely PVL+ (49). In this study, we surveyed and characterized PVL+ strains of MRSA isolated from patients in hospitals. We also examined the in vitro susceptibilities of PVL+ MRSA strains, including those isolated in this study and (with various sequence types) from other countries, to 31 antimicrobial agents, including DX-619, a des-fluoro(6) [des-F(6)] quinolone, and sitafloxacin, a fluoroquinolone.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains. A total of 2,101 MRSA strains isolated from patients in hospitals in Japan from 1996 through 2002 by a levofloxacin surveillance group (led by Keizo Yamaguchi) were examined for PVL genes in this study. CA-MRSA ST30 strains used included SCCmecIVc strains NN1, NN12, NN31A, and NN31D isolated from Japan (48) and HT20030336 isolated from The Netherlands and SCCmecIVa strains USA1100 isolated from the United States (30) and HT20010466 isolated from Australia. PVL+ MRSA ST30 strains 80s-1 (SCCmecIVx; x, subtype unknown), 80s-2 (SCCmecIVc), 80s-3 (SCCmecIVa), 80s-4 (SCCmecIVa), 90s-1 (SCCmecIVx), and 90s-2 (SCCmecIVc) were isolated from "hospital MRSA panics" in the 1980s or 1990s in Japan (49). PVL-positive CA-MRSA strains with sequence types other than ST30 used included strains USA400 (MW2; ST1, SCCmecIVa), USA300 (ST8, SCCmecIVa), and USA1000 (ST59, SCCmecIV) from the United States; strains SSF17 (ST59, SCCmecIV) and Ce7 (ST59) from Taiwan; strain HT20030345 (ST80) from The Netherlands; and strain HT20030442 (ST80) from France. Strains HT20030336, HT20010466, HT20030345, and HT20030442 were kindly provided by Jerome Etienne; strains US1100, USA400, USA300, and USA1000 by L. K. McDougal and L. L. McDonald; and strains SSF17 and Ce7 by Ma Ling and Leung K. Siu as strains representative of different sequence types.

Media and bacterial growth. For bacterial growth, we used LB broth (Difco, Sparks, MD), which was inoculated and incubated at 37°C to log phase with agitation, as the liquid medium. Nutrient agar (Eiken Chemical, Tokyo) was used as the solid medium.

Molecular typing. Multilocus sequence typing (MLST) was performed using seven housekeeping genes, as previously described (13). An allelic profile (allele number) was obtained from the MLST website (http://www.mlst.net/), and the ST data were further analyzed using eBURST software (14) to determine a clonal complex to which each ST belonged. spa (staphylococcal protein A gene) typing was performed as previously described (46). The spa type was determined using a public spa type database (http://tools.egenomics.com/). Detection of the accessory gene regulator (agr) allele group was done by PCR with the reported primers, as previously described (47). The SCCmec types (I to V) were analyzed by PCR as previously described (38) by use of reference strains. In the case of SCCmecIV, three subtypes (IVa, IVb, and IVc) were further analyzed by PCR with the reported primers, as previously described (24).

Virulence gene analysis. Forty-one staphylococcal virulence genes were detected by PCR using the previously reported primers. The targeted genes were three leucocidin genes (26, 34), five hemolysin genes (26), 16 staphylococcal enterotoxin genes (3, 23, 25, 39, 57), one putative staphylococcal enterotoxin gene (28), three exfoliative toxin genes (3, 55), an exotoxin-like gene cluster (54), the epidermal cell differentiation inhibitor gene (26), and 14 adhesin genes (32, 40, 50, 51, 53).

Drug resistance gene analysis. Resistance genes were detected by PCR. They included genes for penicillin resistance (29), aminoglycoside resistance (7, 8, 29), and macrolide and lincosamide resistance (29).

Coagulase typing. MRSA and methicillin-susceptible S. aureus strains were examined for coagulase type by use of a staphylococcal coagulase antiserum kit (Denka Seiken, Tokyo, Japan) in accordance with the manufacturer's instructions.

PFGE and computer analysis. For pulsed-field gel electrophoresis (PFGE) analysis, total bacterial DNA was extracted from MRSA or methicillin-susceptible S. aureus strains and digested with SmaI (21). The digested DNA was applied on PFGE (1.2% agarose). Computer-assisted analysis of the PFGE patterns was performed using a program called Molecular Analyst Finger Printing PLUS (Bio-Rad, Tokyo, Japan), according to a clustering algorithm for the unweighted-pair group method using average linkages (36).

Plasmid analysis. Plasmid DNA was isolated using a QIAGEN Plasmid Midi kit (QIAGEN, Hilden, Germany) and lysostaphin (Wako Pure Chemicals, Osaka, Japan) according to the instructions of the manufacturer. Plasmid DNA was then introduced into S. aureus RN2677 (49) by electroporation using a Gene Pulser II electroporator (Bio-Rad) according to the manufacturer's instructions. Briefly, RN2677 cells were grown at 37°C in brain heart infusion broth (Difco) to log phase (optical density at 600 nm of 0.3). Bacterial cells from 100 ml of culture were harvested, washed with 40 ml of cold sterilized water twice, and suspended in 10 ml of cold 10% (vol/vol) glycerol and finally in 0.5 ml of cold 10% (vol/vol) glycerol. Competent cells (50 µl) thus made were mixed with 1 µl of plasmid DNA, and the mixture was subjected to electroporation in a 0.2-cm electrode spacing cuvette (Bio-Rad) (setting, 100 {Omega}, 2.5 kV, 25 µF). One milliliter of brain heart infusion broth was then added, and the mixture was incubated for 90 min at 37°C. Resistance plasmid-containing clones were selected on agar plates containing cadmium acetate at 10 µg/ml, kanamycin at 10 µg/ml, or erythromycin at 10 µg/ml. Plasmid was isolated using a QIAGEN Plasmid Midi kit (QIAGEN) and lysostaphin (Wako Pure Chemicals) according to the instructions of the manufacturer and analyzed by agarose gel electrophoresis.

Antimicrobial agents. DX-619, a novel des-F(6) quinolone (17), and sitafloxacin, a novel fluoroquinolone (1), were described previously. Other antimicrobial agents were gifts from their manufacturers.

Susceptibility testing. Susceptibility testing of bacterial strains was done by agar dilution method with Mueller-Hinton agar (Difco) according to previously described procedures (9, 35). The final concentrations of antimicrobial agents ranged from 0.001 to 128 µg/ml.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of PVL+ MRSA. Among a total of 2,101 MRSA strains examined, two were PVL+ (0.1%). One strain (strain EB00449) was isolated from the pus of an outpatient (male) aged 27 years in 2002. The other strain (strain DB00319) was isolated from centesis of an inpatient (male) aged 61 years in 2000.

Characteristics of PVL+ MRSA. To investigate the evolutionary relationships and the characteristics of virulence and drug resistance, strains EB00449 and DB00319 were examined for genotypes, virulence genes, penicillinase (PCase) plasmids, and drug resistance genes (Table 1). Strain EB00449 belonged to ST30, while strain DB00319 possessed a novel sequence type (ST765, a single-locus variant of the founder [ST30] of clonal complex 30 [CC30]). The two strains were also distinguishable from each other in terms of spa type, i.e., spa type 19 for EB00449 and spa type 43 for DB00319. In addition, EB00449 was negative for hlb (which codes for ß-hemolysin), sea (which codes for staphylococcal enterotoxin A), and icaA (which codes for biofilm formation), while DB00319 was positive for hlb, sea, and icaA.


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  		TABLE 1. Characteristics of PVL+ MRSA strains isolated in this study compared with characteristics of nosocomial-outbreak-derived PVL+ MRSA ST30 strains and PVL+ CA-MRSA ST30 strains from Japan and CA-MRSA ST30 strains from other countriesa

 
Moreover, EB00449 was resistant only to ß-lactam antimicrobial agents and carried a 32-kb simple PCase plasmid coding for resistance to ampicillin (and penicillin G) and cadmium (Table 1; Fig. 1); EB00449 carried an additional, 46-kb plasmid of unknown function (Fig. 1). In contrast, DB00319 was resistant to multiple drugs and carried a 44-kb multidrug-resistant PCase plasmid coding for resistance not only to ampicillin (and penicillin G) and cadmium but also to kanamycin, streptomycin, erythromycin, and clindamycin (Table 1; Fig. 1); DB00319 carried an additional, 5.4-kb plasmid of unknown function (Fig. 1).


Figure 1
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  		FIG. 1. Plasmid analysis of PVL+ MRSA strains DB00319 and EB00449 and S. aureus RN2677 carrying a PCase plasmid. Plasmid DNA from each bacterial strain was electrophoresed in 0.7% agarose. Lanes: 1, strain DB00319; 2, RN2677 carrying a PCase plasmid (pAC00s7) from strain DB00319; 3, strain EB00449; 4, RN2677 carrying a PCase plasmid (pAC00s8) from strain EB00449; 5, RN2677 (strain without plasmid). Plasmid sizes were determined by using reference plasmids with known molecular sizes. The plasmid sizes thus determined are indicated to the left of the gel.

 
The data for strains EB00449 and DB00319 were compared with data for PVL+ MRSA strains (strains 80s-1 to -4 and 90s-1 and -2) isolated during the nosocomial outbreaks in the 1980s and 1990s, recent PVL+ CA-MRSA strains (strains NN1 and NN12) from Japan, and PVL+ CA-MRSA ST30 strains from other countries (Table 1). Strain EB00449 was very similar to PVL+ CA-MRSA strain NN1 from Japan; there was a divergence in that EB00449 carried an additional 46-kb plasmid compared with NN1, which carried only PCase plasmid. Moreover, DB00319 closely resembled nosocomial-outbreak-derived PVL+ HA-MRSA strains, especially strain 90s-1, except for the sequence type.

Cluster analysis of PFGE patterns. A computer-assisted comparison of PFGE patterns obtained with PVL+ HA-MRSA strains (80s-1 to -4 and 90s-1 and -2) isolated during the nosocomial outbreaks in the 1980s and 1990s, strains DB00319 and EB00449, recent PVL+ CA-MRSA strains (NN1 and NN12) from Japan, and PVL+ CA-MRSA ST30 strains from other countries is shown in Fig. 2. Consistent with the spa typing, PVL+ HA-MRSA and DB00319 (spa type 43) strains constituted subclusters, which were distinguished from subclusters of PVL+ CA-MRSA and EB00449 (spa type 19 or 654) strains.


Figure 2
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  		FIG. 2. PFGE analysis of PVL+ MRSA strains (DB00319 and EB00449) isolated in this study, nosocomial-outbreak-derived PVL+ MRSA ST30 strains (80s-1 to -4 and 90s-1 to -2) isolated in the 1980s and 1990s, PVL+ CA-MRSA ST30 strains (NN1 and NN12) from Japan, and PVL+ CA-MRSA ST30 strains (USA1100, HT20030336, and HT20010466) from other countries (right side) and dendrogram constructed by computer-assisted comparison (left side). The characteristics of each bacterial strain are listed in Table 1.

 
Characteristics of PVL+ MRSA strains not of ST30. Characteristics of global PVL+ MRSA strains belonging to U.S. sequence types (ST1 and ST8), a Taiwanese (or U.S.) sequence type (ST59), and a European sequence type (ST80) are summarized in Table 2. A remarkable difference in adhesive properties (genes) was observed between strains of ST30 and strains not of ST30. In contrast to ST30 strains, strains not of ST30 were negative for bbp (which codes for bone sialoprotein adhesin). Moreover, many strains not of ST30 (except for the U.S. ST1 strain) were negative for cna (which codes for collagen adhesin).


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  		TABLE 2. Characteristics of global PVL+ MRSA strains not of ST30

 
Regarding toxin genes, the U.S. strains (ST1 and ST8) were positive for sea, just like nosocomial-outbreak-derived PVL+ HA-MRSA strains from Japan. U.S. ST1 and European ST80 strains, but not ST8, ST30, or ST59 strains, were positive for seh.

In vitro susceptibility. Next, PVL+ MRSA strains (22 strains) were examined for susceptibility to 31 antimicrobial agents. They included the two strains DB00319 (ST765) and EB00449 (ST30), six PVL+ HA-MRSA (ST30) strains isolated from nosocomial outbreaks in the 1980s and 1990s, four PVL+ CA-MRSA (ST30) strains from Japan, and 10 PVL+ CA-MRSA (ST1, ST8, ST30, ST59, and ST80) strains from other countries. The data are summarized in Table 3.


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  		TABLE 3. In vitro activities of DX-619 and other antimicrobial agents against PVL+ MRSA strainsa

 
DX-619 showed the greatest activity among the antimicrobial agents tested (MIC50, 0.008 µg/ml; MIC90, 0.008 µg/ml). This activity was followed by those of rifampin (MIC50, 0.008 µg/ml; MIC90, 0.016 µg/ml) and sitafloxacin or tosufloxacin (MIC50, 0.031 µg/ml; MIC90, 0.063 µg/ml). Sitafloxacin showed a better MIC range than tosufloxacin (0.016 to 0.125 µg/ml versus 0.016 to 0.25 µg/ml, respectively).

Gentamicin resistance (MICs, 8 to ≥256 µg/ml) was found only in Japanese PVL+ MRSA ST30 strains (in two of four PVL+ CA-MRSA strains, in five of six nosocomial-outbreak-derived PVL+ HA-MRSA strains, and in one [strain DB00319] of two PVL+ MRSA strains isolated in this study) (Tables 1, Table 2, and Table 3). Minocycline resistance (MIC, 8 µg/ml) was also found only in Japanese PVL+ MRSA ST30 strains (nosocomial-outbreak-derived PVL+ HA-MRSA strain 80s-1) (Table 1, Table 2 and Table 3). Fusidic acid resistance (MIC, 8 µg/ml) was found only in European PVL+ CA-MRSA ST80 strains (Table 1, Table 2, and Table 3).

Resistance to some other drugs was distributed among different sequence types: kanamycin resistance (MICs, ≥256 µg/ml) among ST8, ST30, ST59, and ST80; streptomycin resistance (MICs, 128 to ≥256 µg/ml) among ST30, ST59, and ST80; erythromycin resistance (MICs, ≥256 µg/ml) among ST8, ST30, and ST59; clindamycin resistance (MICs, ≥256 µg/ml) among ST30 and ST59; and tetracycline resistance (MICs, ≥16 µg/ml) among ST1, ST8, ST30, ST59, and ST80.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PVL+ CA-MRSA strains in Japan are of a worldwide sequence type (ST30) (48). No cases of ST1 and ST8, found mainly in the United States (30, 52), ST80, found mainly in Europe (52), or ST59, found mainly in Taiwan and the United States (4, 12), have been found in Japan.

There were nosocomial MRSA outbreaks ("hospital MRSA panics") in Japan in the late 1980s and early 1990s. At that time, decubitus, pneumonia, bacteremia, and postoperative MRSA enteritis were found frequently (18, 27, 45), but they have drastically decreased recently. Interestingly, MRSA strains isolated during the nosocomial outbreaks were largely PVL+ (49). Although these PVL+ HA-MRSA strains belonged to ST30, similarly to present PVL+ CA-MRSA strains, they had unique features (49), e.g., they exhibited spa type 43, possessed hlb, sea (which is associated with severity of infection [sepsis and shock] [15]), and icaA, were multidrug resistant, and carried a multidrug-resistant PCase plasmid, in contrast to present PVL+ CA-MRSA ST30 strains (spa type 19). The possibility of high virulence of hlb+ sea+ multidrug-resistant PVL+ HA-MRSA strains remains to be clarified.

In this study, we further surveyed PVL+ MRSA strains from patients in hospitals, and two PVL+ MRSA strains were studied. One PVL+ MRSA strain, strain DB00319, from an inpatient aged 61 years, shared common features (CC30, spa type 43, hlb, sea, icaA, and multidrug-resistant PCase plasmid) with multidrug-resistant PVL+ HA-MRSA strains from the "hospital MRSA panics" but with a genetic divergence in sequence type (novel ST765). ST765 is a single-locus variant (single base substitution in tpi) of the founder type, ST30, of CC30. This suggests that a "hospital MRSA panic" clone continues to exist in hospitals in Japan, with a slight divergence.

The other PVL+ MRSA strain, strain EB00449, from an outpatient aged 27 years, was a PVL+ CA-MRSA ST30 strain. EB00449 was, however, slightly divergent in terms of plasmid profile from previously characterized PVL+ CA-MRSA ST30 strains, i.e., strain NN1, isolated from an infant (aged 11 months) with bullous impetigo (48), and strain NN12, carrying a conjugative drug resistance plasmid, isolated from a high school student (a basketball player aged 18 years) with bacteremia and osteomyelitis (40, 48). Thus far, PVL+ CA-MRSA ST30 strains found in Japan have been distributed among individuals below 30 years of age.

Both types of PVL+ MRSA strains of CC30 present in Japan, PVL+ hlb+ sea+ HA-MRSA ST30/765 spa type 43 strains (continuing from the 1980s) and PVL+ CA-MRSA spa type 19 strains (emerged in the community recently), are highly adhesive (bbp+ and cna+). bbp codes for bone sialoprotein adhesin, which leads to hematogenously spread osteomyelitis and arthritis (20, 42, 43). cna codes for collagen adhesin, which is correlated with pulmonary manifestations (22). Such highly adhesive characteristics may be a cause of continuous infection in hospitals and in the community in Japan.

Twenty-two PVL+ MRSA strains, including PVL+ HA-MRSA ST30/765 strains and recent PVL+ CA-MRSA ST30 strains from Japan and PVL+ CA-MRSA strains with various global sequence types (ST1, ST8, ST30, ST59, and ST80) from other countries, were examined for their susceptibilities to 31 antimicrobial agents. Some drug resistance is associated with sequence type, e.g., fusidic acid resistance with ST80 (52). In Japan, gentamicin has been widely used not only in hospitals but also in the community as an ointment for skin infection, and due to this, gentamicin-resistant PVL+ MRSA ST30/765 strains are notable.

Antimicrobial agents tested in this study included DX-619, a des-F(6) quinolone, which has shown an in vitro activity against gram-positive bacteria (17), and sitafloxacin, a fluoroquinolone (1). DX-619 showed the greatest activity against PVL+ MRSA among the antimicrobial agents tested (MIC90, 0.008 µg/ml; MIC50, 0.008 µg/ml). This activity was eightfold greater than that of sitafloxacin (MIC90, 0.063 µg/ml), 32-fold greater than that of levofloxacin (MIC90, 0.25 µg/ml), and 125-fold greater than that of vancomycin (MIC90, 1 µg/ml). It was even twofold greater than that of rifampin (MIC90, 0.016 µg/ml). Among fluoroquinolones tested, sitafloxacin showed the greatest activity, e.g., it showed a better MIC range than tosufloxacin (0.016 to 0.125 µg/ml versus 0.016 to 0.25 µg/ml, respectively).

The effects of DX-619 (or sitafloxacin) on severe diseases due to PVL+ CA-MRSA strains (such as bacteremia, osteomyelitis, or necrotizing pneumonia) remain to be investigated.

In conclusion, there exist two types of highly adhesive (bbp+ cna+) PVL+ MRSA strains in Japan: hlb+ sea+, multidrug-resistant HA-MRSA ST30/765 strains (spa type 43) (originating from "hospital MRSA panics") and CA-MRSA ST30 strains (spa type 19) carrying or not carrying the conjugative drug resistance plasmid (emerged recently in the community). DX-619 showed higher activity than other anti-MRSA agents against those PVL+ MRSA strains as well as global PVL+ CA-MRSA strains with ST1, ST8, ST30, ST59, and ST80. Further studies are necessary to evaluate the anti-PVL+ MRSA activity of DX-619 (or sitafloxacin) in vivo, such as in animal infection models.


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   ACKNOWLEDGMENTS
 
We thank Jerome Etienne, L. K. McDougal, L. L. McDonald, Ma Ling, and Leung K. Siu for PVL+ MRSA strains.

This study was supported by a grant from Daiichi Pharmaceutical Co., Ltd., Tokyo, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, 757 Ichibanchou, Asahimachidori, Niigata 951-8510, Japan. Phone: (81) (25) 227-2050. Fax: (81) (25) 227-0762. E-mail: tatsuoy{at}med.niigata-u.ac.jp. Back

{triangledown} Published ahead of print on 16 October 2006. Back


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 Results
 Discussion
 References
 

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Antimicrobial Agents and Chemotherapy, December 2006, p. 4077-4086, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00847-06
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