Infrequent occurrence of single mutations in topoisomerase IV and DNA gyrase genes among US levofloxacin-susceptible clinical isolates of Streptococcus pneumoniae from nine institutions (1999-2003)

OBJECTIVES: Prevalence of single quinolone-resistance determining region (QRDR) mutations in Streptococcus pneumoniae was studied from nine institutions over 5 years to track the incidence of single QRDR mutations. METHODS: All 1106 levofloxacin-susceptible pneumococci (MICs < or = 2.0 mg/L) identified from 1112 total isolates (99.5% susceptibility) in TRUST 3 (1999), TRUST 5 (2001) and TRUST 7 (2003) surveillance studies from the same nine hospitals in nine states were screened for QRDR mutations. Using pyrosequencing, the strains were screened for mutations corresponding to hot spots Asp-78, Ser-79 and Asp-83 in ParC; Asp-80, Ser-81 and Glu-85 in GyrA; Asp-435 in ParE and Asp-435 in GyrB. DNA sequencing of QRDRs was performed to confirm mutations. RESULTS: No QRDR mutations were found in any of the isolates with levofloxacin MICs < or = 0.5 mg/L and no gyrA or gyrB QRDR mutations were found in any of the screened isolates (MICs < or = 2 mg/L). Four single-step QRDR mutants with the following amino acid substitutions were found: ParE Asp-435 to Asn (isolated in 1999 in Colorado); ParC Asp-83 to Asn (isolated in 2001 in Kentucky); ParC Ser-79 to Phe (isolated in 2003 in Indiana) and ParC Ser-79 to Tyr (isolated in 2003 in California). These non-clonal strains had levofloxacin MICs of 1 mg/L and were non-susceptible to ciprofloxacin (MIC 2-4 mg/L). CONCLUSIONS: Overall prevalence of single QRDR mutations in levofloxacin-susceptible S. pneumoniae with MICs of < or = 2 mg/L was 0.4% (4/1106) and has remained <1% within nine institutions over 5 years (1999-2003).     

Increasing resistance of Streptococcus pneumoniae to fluoroquinolones: results of a Hong Kong multicentre study in 2000.

The MICs of 13 antimicrobial agents including seven fluoroquinolones (ciprofloxacin, levofloxacin, sparfloxacin, grepafloxacin, gatifloxacin, moxifloxacin and clinafloxacin) for Streptococcus pneumoniae isolates obtained from all regions of Hong Kong in the year 2000 were determined by the Etest. Overall, 39.4% of 180 isolates were susceptible to penicillin, 11.7% were intermediate and 48.9% were resistant. The overall prevalence of fluoroquinolone non-susceptibility (levofloxacin MIC > or = 4 mg/L) was 13.3% but increased to 27.3% among the penicillin-resistant isolates. For the fluoroquinolone non-susceptible isolates, within-class cross-resistance was common. For the fluoroquinolone non-susceptible isolates, the median MICs of clinafloxacin, gatifloxacin, moxifloxacin, sparfloxacin and grepafloxacin were, respectively, six-, 24-, 32- 84- and 128-fold higher than those for the susceptible isolates. All fluoroquinolone non-susceptible strains were derived from adults. The prevalence of fluoroquinolone resistance was higher in isolates from older patients (17.1% among those > or = 65 years of age versus 9.1% among those 18-64 years of age, P < 0.001) and from adults with chronic obstructive pulmonary disease (24.6% versus 9.3%, P = 0.01). All fluoroquinolone non-susceptible strains were non-susceptible to penicillin (MIC range 2-4 mg/L), cefotaxime (MIC range 1-4 mg/L) and erythromycin (MIC range 4- > or = 256 mg/L). The fluoroquinolone non-susceptible isolates were genetically related to the Spain(23F)-1 clone when analysed by pulse-field gel electrophoresis and multilocus sequence typing. In conclusion, a rapid increase in the prevalence of fluoroquinolone resistance among S. pneumoniae was found in Hong Kong. Typing analysis suggests that this is due to the pan-regional dissemination of a fluoroquinolone-resistant variant (designated Hong Kong(23F)-1) of the globally distributed Spain(23)F-1 clone.     

Emergence and epidemiology of fluoroquinolone-resistant Streptococcus pneumoniae strains from Italy: report from the SENTRY Antimicrobial Surveillance Program (2001-2004)

Fluoroquinolones are key antimicrobials in the treatment of more serious pneumococcal infections, especially for treating infections caused by penicillin-resistant strains. Increased use of newer fluoroquinolones should be accompanied by greater surveillance efforts to monitor resistance development as well as clonal dissemination. Streptococcus pneumoniae (n=551) collected from 3 medical centers in Italy (Catania, Genoa, and Rome) over a period of 4 years (2001-2004) as part of the SENTRY Antimicrobial Surveillance Program were susceptibility tested against >30 antimicrobials using reference broth microdilution methods. Mutations in the quinolone resistance-determining region (QRDR) were characterized by PCR and sequencing of parC, parE, and gyrA. Epidemiological relationships among levofloxacin-resistant isolates were determined using ribotyping, PFGE, serotyping combined with antimicrobial resistance profiles augmented by QRDR mutation patterns. Eighty-three (15.1%) isolates showed reduced susceptibility to ciprofloxacin (MIC>or=4 microg/mL) and 31 (5.6%) were resistant to levofloxacin. In 2001, all pneumococcal isolates were susceptible to levofloxacin and resistance rapidly emerged in all 3 medical centers in 2002. The overall rates of levofloxacin resistance in 2002-2004 were the following: Catania 10.9%, Genoa 3.3%, and Rome 6.5%. All resistant strains showed at least one mutation in parC and gyrA. Each isolate from Genoa with a unique resistance phenotype also showed distinct ribotype/PFGE, serotype, and QRDR mutation patterns. All isolates from Catania (n=19) showed an identical ribotype/PFGE pattern (333.3/A1); however, 3 distinct clusters could be identified based on further resistance phenotype, serotypes, and QRDR mutation pattern analysis. Two clusters were documented among isolates from Rome based on ribotype/PFGE. One isolate from Genoa shared ribotype/PFGE (333.3/A1) and serotype (9 not V) results with clusters from the other 2 institutions monitored, indicating clonal dissemination between the geographically diverse cities. In conclusion, fluoroquinolone resistance rates have increased among S. pneumoniae recovered in Italian medical centers evaluated by the SENTRY Program. Although resistance has emerged in many epidemiologically distinct strains, clonal dissemination seems to be a key contributing factor for increasing resistance to fluoroquinolones among pneumococci in this nation.     

Spain14-5 international multiresistant Streptococcus pneumoniae clone resistant to fluoroquinolones and other families of antibiotics

The Spain(14)-5 international multiresistant clone was initially described as resistant to penicillin, tetracycline, chloramphenicol and trimethoprim/sulfamethoxazole. In Gipuzkoa, Spain, Streptococcus pneumoniae isolated from 16 patients, and determined by PFGE and multilocus sequence typing to belong to the Spain(14)-5 clone, showed further resistance to fluoroquinolones (all strains had point mutations in the parC and gyrA genes). In addition, most strains showed resistance to amoxicillin (MIC > or = 8 mg/L), cefotaxime (MIC > or = 2 mg/L), macrolides and lincosamides. Two strains were resistant to rifampicin (MIC 8 mg/L). The multiresistance observed in these isolates converts the Spain(14)-5 clone into one of the most, if not the most, multiresistant of the international clones described.     

Characterization of fluoroquinolone-resistant beta-hemolytic Streptococcus spp. isolated in North America and Europe including the first report of fluoroquinolone-resistant Streptococcus dysgalactiae subspecies equisimilis: report from the SENTRY Antimicrobial Surveillance Program (1997-2004)

Beta-hemolytic streptococci are common bacterial pathogens that can cause serious invasive disease, and although this group of species has remained susceptible to the fluoroquinolone class, resistant strains have been reported. This multicenter investigation determined the rate of fluoroquinolone-resistant beta-hemolytic streptococci using the SENTRY Antimicrobial Surveillance Program network data (1997-2004). Forty-seven surveillance culture isolates of beta-hemolytic streptococci from North America and Europe with elevated levofloxacin MIC results (2 to >32) microg/mL were tested for susceptibility to other fluoroquinolones including ciprofloxacin, garenoxacin, gatifloxacin, gemifloxacin, and moxifloxacin using reference broth microdilution and Etest (BIODISK, Solna, Sweden) methods. Strains were characterized using polymerase chain reaction and sequencing to detect mutations in the quinolone-resistance determining region (QRDR). The beta-hemolytic streptococci isolates with reduced fluoroquinolone susceptibility included the following Lancefield groups: A (Streptococcus pyogenes; 9 strains), B (Streptococcus agalactiae; 24 strains), C and G (14 strains). Vitek and API 20 strep (bioMerieux, Hazelwood, MO) identification systems, as well as conventional biochemical methods and colony morphology, were used to confirm the organism identifications. The overall potency (MIC90 in microg/mL) for the fluoroquinolones against all tested beta-hemolytic streptococci showed the following rank order: gemifloxacin (0.06) > garenoxacin (0.12) > moxifloxacin (0.25) > gatifloxacin (0.5) > levofloxacin = ciprofloxacin (1). The rate of levofloxacin-resistant beta-hemolytic streptococci in the SENTRY program was 0.14% (Europe) and 0.51% (North America) during the study period. All levofloxacin-resistant strains tested by molecular methods had significant mutations in either parC (position 79 or 83) and/or gyrA (position 81 or 85). All but 2 isolates with high-level resistance to levofloxacin (>32 microg/mL) had gyrA mutations. Strains with lower MIC values to levofloxacin (2-4 microg/mL) only had mutations in parC. The increasing rate of fluoroquinolone-resistant streptococci including Streptococcus pneumoniae, viridans group streptococci, and the more recently reported beta-hemolytic streptococci, is becoming a clinical concern due to the morbidity and mortality caused by these pathogens.     

Prevalence of Single Mutations in Topoisomerase Type II Genes among Levofloxacin-Susceptible Clinical Strains of Streptococcus pneumoniae Isolated in the United States in 1992 to 1996 and 1999 to 2000

Levofloxacin resistance in Streptococcus pneumoniae is rare, requiring at least two mutations in the quinolone resistance-determining region (QRDR) of topoisomerase IV and DNA gyrase. The prevalence of single QRDR mutations in these genes is unknown. Of 9,438 levofloxacin-susceptible pneumococci from the TRUST 4 surveillance study (1999-2000), 528 strains (MICs of 0.5 to 2.0 microg/ml) were selected for analysis. For comparison, 214 levofloxacin-susceptible strains (MICs of 0.5 to 1 microg/ml) isolated between 1992 and 1996 were analyzed. Oligonucleotide probe assay and DNA sequencing were used to detect QRDR mutations leading to changes at Ser79 and Asp83 in ParC, Ser81 in GyrA, and Asp435 in ParE, the most frequently found substitutions among levofloxacin-resistant strains. Among the 1992 to 1996 isolates only one strain (levofloxacin MIC, 1 microg/ml) had a mutation (Ser79 to Phe in ParC). No single mutations were found among 270 TRUST 4 strains with levofloxacin MICs of 0.5 microg/ml. Among 244 strains for which levofloxacin MICs were 1 microg/ml, 15 strains (6.1%) had a parC mutation and 3 strains (1.2%) had a parE mutation. Of 14 strains for which levofloxacin MICs were 2 microg/ml, 10 strains (71%) had a parC mutation; no parE mutations were found. No gyrA mutations were detected. It was estimated that 4.5% of the 9,438 levofloxacin-susceptible TRUST 4 isolates (MICs, < or =0.06 to 2 microg/ml) had a single parC or parE QRDR mutation. Although there has been an increase in the prevalence of single-step mutants, the increase may have been overestimated due in part to differences in geographical distribution for the two sets of isolates.     

Levofloxacin-resistant invasive Streptococcus pneumoniae in the United States: evidence for clonal spread and the impact of conjugate pneumococcal vaccine

The emergence of fluoroquinolone resistance in sterile-site isolates of Streptococcus pneumoniae is documented in this study characterizing all invasive levofloxacin-resistant (MIC, > or = 8 mg/liter) S. pneumoniae isolates (n = 50) obtained from the Centers for Disease Control and Prevention Active Bacterial Core Surveillance from 1998 to 2002. Resistance among all isolates increased from 0.1% in 1998 to 0.6% in 2001 (P = 0.008) but decreased to 0.4% in 2002, while resistance among vaccine serotypes continued to increase from 0.3% in 1998 to 1.0% in 2002, suggesting that fluoroquinolones continue to exert selective pressure on these vaccine serotypes. Only 22% of resistant isolates were not covered by the conjugate vaccine serogroups. Multilocus sequence typing revealed that 58% of resistant strains were related to five international clones identified by the Pneumococcal Molecular Epidemiology Network, with the Spain(23F)-1 clone being most frequent (16% of all isolates). Thirty-six percent of the isolates were coresistant to penicillin, 44% were coresistant to macrolides, and 28% were multiresistant to penicillin, macrolides, and fluoroquinolones. Fifty percent of the isolates were resistant to any three drug classes. Ninety-four percent of the isolates had multiple mutations in the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes. In 16% of the isolates, there was evidence of an active efflux mechanism. An unusual isolate was found that showed only a single parE mutation and for which the ciprofloxacin MIC was lower (2 mg/liter) than that of levofloxacin (8 mg/liter). Our results suggest that invasive pneumococcal isolates resistant to levofloxacin in the United States show considerable evidence of multiple resistance and of clonal spread.     

上海地区肺炎链球菌对氟喹诺酮类的敏感性及其喹诺酮耐药决定区突变研究

目的了解上海地区临床分离肺炎链球菌对氟喹诺酮类等抗菌药物的敏感性，并对氟喹诺酮类敏感菌株的喹诺酮耐药决定区（QRDR）突变进行初步研究。方法收集上海地区部分医院2004-2005年共176株肺炎链球菌临床分离株，用琼脂稀释法测定环丙沙星、左氧氟沙星、加替沙星和莫西沙星等15种抗菌药物的抗菌活性，并选取部分左氧氟沙星敏感肺炎链球菌菌株进行QRDRPCR扩增、测序。结果176株受试菌株中．PSSP、PISP和PRSP各占48．9％、47．1％和4．0％，受试菌株对环丙沙星、左氧氟沙星、加替沙星和莫西沙星敏感率均为100％。QRDR的扩增测序结果发现，20株左氧氟沙星MIC1～2mg／L的敏感菌株中，19株的parC、parE基因上已存在不同程度的氨基酸突变．包括ParC：Phe 105→Leu／Asp136→Tyr，Pare：Asp435→AsnjIle460→ValjAsn477→Lys．而在gyrA、gyrB基因上仅发现点突变，未导致相关氨基酸突变。结论上海地区未发现氟喹诺酮类耐药肺炎链球菌临床分离株，但左氧氟沙星MIC1～2mg／L的敏感株中已发现QRDR一级突变现象，突变的基因位点均见于parC、pare基因。     

Target site modifications and efflux phenotype in clinical isolates of Streptococcus pneumoniae from Hong Kong with reduced susceptibility to fluoroquinolones

Ciprofloxacin-susceptible (n = 7) and -resistant (MIC >or=4 mg/L) (n = 15) clinical isolates of Streptococcus pneumoniae from diverse sources in Hong Kong were studied for target site modifications and efflux phenotype. Reserpine-inhibited efflux of ciprofloxacin and/or levofloxacin was common in both susceptible and non-susceptible isolates. The ParC substitutions K137N and/or S79F or Y were associated with increased ciprofloxacin MICS. The GyrA substitution S81F was only found in isolates with full resistance to ciprofloxacin (MIC >or=16 mg/L) and levofloxacin (MIC >or=8 mg/L). Among clinical isolates of S. pneumoniae, accumulation of target site mutations in strains with an efflux mechanism was associated with increasing MICs of fluoroquinolones.     

Clonal spread of fluoroquinolone non-susceptible Streptococcus pyogenes

BACKGROUND: Fluoroquinolones are an important group of antibiotics widely used in adults, and, despite the absence of official approval, these drugs are also used in children. So far, resistance to fluoroquinolones in Streptococcus pyogenes is very rare. METHODS: During a national surveillance programme in Belgium from 1999 to 2002, 2793 non-duplicate S. pyogenes recovered from tonsillopharyngitis patients were screened for fluoroquinolone resistance. Mutations in topoisomerase genes and the presence of any efflux pump activity were investigated to elucidate the fluoroquinolone resistance mechanisms. Clonality was assessed by pulsed-field gel electrophoresis (PFGE) and emm typing. RESULTS: Non-susceptibility to fluoroquinolones, defined as ciprofloxacin MIC > or = 2 mg/L, was identified in 152 (5.4%) of 2793 S. pyogenes. Fifty-five (36%) fluoroquinolone non-susceptible isolates were investigated for known resistance mechanisms; all showed mutations in parC, and 29 (19%) isolates also in parE; antibiotic efflux was not noted. Two major PFGE types comprised 88% of fluoroquinolone non-susceptible S. pyogenes and belonged to serotypes emm6 and emm75. Overall, emm6 and emm75 constituted >90% of all fluoroquinolone non-susceptible isolates and showed a significant temporal and geographical shift within Belgian provinces. Although fluoroquinolone-susceptible S. pyogenes also showed fluctuations in the predominant S. pyogenes serotypes, emm6 or emm75 were under-represented in this population. Approx. 55% of the fluoroquinolone non-susceptible isolates were recovered from children ( < or =16 years). CONCLUSIONS: We show here, for the first time, a multi-clonal spread of fluoroquinolone non-susceptible S. pyogenes exhibiting a known resistance mechanism. Non-susceptibility to fluoroquinolones in paediatric isolates is of concern.     

In vitro activity of telithromycin against respiratory tract pathogens in comparison with other antimicrobial agents

This study was done to evaluate the in vitro activity of a new ketolide telithromycin in comparison with clarithromycin, erythromycin, moxifloxacin and levofloxacin against Streptococcus pneumoniae (n = 67), Haemophilus influenzae (n = 139), and Moraxella catarrhalis (n = 46)collected between January and June 2003 in Hong Kong. Among the H. influenzae isolates, 25.2% produced beta-lactamase, while 97.8% of M. catarrhalis isolates produced beta-lactamase. Half of the S. pneumoniae isolates were nonsusceptible to penicillin, and 90.9% of these strains were resistant to clarithromycin and erythromycin. One (1.5%) S. pneumoniae strain was resistant to levofloxacin (MIC = 8 mg/l) and all isolates were sensitive to moxifloxacin and telithromycin with MIC <1 mg/l. H. influenzae isolates were sensitive to all fluoroquinolones tested and 2.2% of H. influenzae were resistant to clarithromycin. M. catarrhalis isolates were sensitive except 1 strain which was resistant to levofloxacin (MIC = 4 mg/l) and moxifloxacin (8 mg/l). All M. catarrhalis strains were sensitive to telithromycin with MIC90 = 0.5 mg/l. Telithromycin demonstrated high activity and no resistance was found in all these major respiratory tract pathogens.     

Worldwide incidence, molecular epidemiology and mutations implicated in fluoroquinolone-resistant Streptococcus pneumoniae: data from the global PROTEKT surveillance programme

OBJECTIVES: To analyse the mutations and epidemiology associated with fluoroquinolone-resistant pneumococci collected as part of the PROTEKT global surveillance programme during 1999-2000. METHODS: Sixty-nine centres in 25 countries submitted a total of 3362 Streptococcus pneumoniae isolates, for which the MICs of antimicrobial agents were determined using NCCLS methodology. RESULTS: Levofloxacin resistance was low overall (1% worldwide), with higher rates in: Hong Kong (14.3%), South Korea (2.9%), USA (1.8%), Mexico (1.5%), Canada (1.4%) and Japan (1.3%). Levofloxacin resistance was very low or absent in European countries, and absent in Australia. Worldwide, there was a total of 35 levofloxacin-resistant isolates, of which 22 (63%) were resistant and 10 (29%) were intermediate to moxifloxacin. All levofloxacin-resistant isolates were susceptible to telithromycin (< or =0.5 mg/L), linezolid (< or =2 mg/L) and quinupristin/dalfopristin (< or =1 mg/L). One or more mutations in the topoisomerase genes were identified in all levofloxacin-resistant isolates; most of these isolates (33/35) had a mutation in one of the DNA gyrase encoding genes (gyrA, gyrB) and one of the topoisomerase IV encoding genes (parC, parE). Eighteen (51%) isolates carried the same combination of amino acid substitutions: Ser-81-->Phe in GyrA and Ser-79-->Phe in ParC. Isolates displaying a levofloxacin MIC of 2-4 mg/L generally had no mutation or one mutation in either a DNA gyrase or a topoisomerase IV gene, although most mutations were in parC. CONCLUSIONS: Most levofloxacin-resistant isolates possess two mutations (one in DNA gyrase and one in topoisomerase IV). Although multilocus sequence typing demonstrated that most of these isolates were unrelated, 12 (34%) were the Spain23F-1 clone: 10 from Hong Kong and one each from Saskatchewan, Canada and Sao Paulo, Brazil.     

Characterization of fluoroquinolone and carbapenem susceptibilities in clinical isolates of levofloxacin-resistant Pseudomonas aeruginosa

BACKGROUND: Pseudomonas aeruginosa can rapidly acquire resistance to antibiotics, including fluoroquinolones and carbapenems. METHODS: We characterized fluoroquinolone, carbapenem and other beta-lactam susceptibilities and analyzed fluoroquinolone and carbapenem resistance in 16 clinical isolates of levofloxacin-resistant P. aeruginosa. RESULTS: All levofloxacin-resistant isolates showed high MICs (> or =32 microg/ml) for fluoroquinolones including norfloxacin, levofloxacin, sparfloxacin, gatifloxacin and pazufloxacin, whereas the MICs for sitafloxacin were between 2 and 16 microg/ml. These isolates had both a Thr83Ile mutation in GyrA and a Ser87Leu mutation in ParC. An additional mutation, Glu469Asp in GyrB, was detected in 3 isolates. Three of 16 isolates found during antibiotic therapy showed resistance to carbapenems (MIC, 16-32 microg/ml) because of a reduced production of OprD. Fluoroquinolones, beta-lactams and sulfamethoxazole-trimethoprim were used for 3 months before the isolation of levofloxacin-resistant P.aeruginosa. CONCLUSIONS: Emergence of resistant isolates to both fluoroquinolones and carbapenems during antibiotic therapy is a serious clinical problem. Our results suggest that susceptibilities to fluoroquinolones as well as carbapenems should be monitored during a prolonged course of antibiotic therapy against P. aeruginosa infection.     

Activity of garenoxacin, an investigational des-F(6)-quinolone, tested against pathogens from community-acquired respiratory tract infections, including those with elevated or resistant-level fluoroquinolone MIC values

Garenoxacin, a novel des-F(6)-quinolone, was tested against 40423 pathogenic isolates associated with community-acquired respiratory tract infections (CA-RTIs). The strains included Streptococcus pneumoniae (18887), Haemophilus influenzae (15555), and Moraxella catarrhalis (5981), each isolated from a significant infection monitored by the SENTRY Antimicrobial Surveillance Program (1999-2005; North America, Latin America, and Europe). All tests were performed by reference broth microdilution methods for garenoxacin and 19 comparison agents. The garenoxacin MIC(90) and percentage (%) of strains inhibited at < or =1 microg/mL (proposed susceptible breakpoint) were S. pneumoniae (0.06 microg/mL, >99.9% susceptible), H. influenzae (< or =0.03 microg/mL, >99.9%), and M. catarrhalis (< or =0.03 microg/mL, 100.0%). The garenoxacin potency versus the pneumococci was 16- to 32-fold greater than levofloxacin or ciprofloxacin and 2-fold superior to moxifloxacin (MIC(90), 0.12 microg/mL). Resistances to other classes of antimicrobials did not adversely influence garenoxacin MIC results. Ciprofloxacin- or levofloxacin-resistant (MIC, > or =4 microg/mL) S. pneumoniae had higher garenoxacin MIC(90) values (1 microg/mL), but 90.6% to 97.5% of strains remained susceptible. Strains of all 3 monitored pathogens with mutations in the quinolone resistance determining region (QRDR) had higher garenoxacin MIC results, with > or =3 to 4 QRDR mutations required to elevate garenoxacin MIC values to > or =2 microg/mL. In conclusion, garenoxacin possesses a potent activity against pneumococci, H. influenzae, and M. catarrhalis strains worldwide, at a level significantly greater than the available tested agents in the fluoroquinolone class (ciprofloxacin, levofloxacin, and moxifloxacin). Only 13 and 4 isolates (0.07% and 0.03%) of S. pneumoniae and H. influenzae, respectively, had a garenoxacin MIC at > or =2 microg/mL, thus, making this new "respiratory antipneumococcal" quinolone an attractive candidate for the therapy of contemporary CA-RTI (bronchitis, pneumonia, and sinusitis).     

In vitro activity of sitafloxacin against clinical strains of Streptococcus pneumoniae with defined amino acid substitutions in QRDRs of gyrase A and topoisomerase IV

OBJECTIVES: Fluoroquinolone-resistant Streptococcus pneumoniae are increasing worldwide rapidly. In vitro activities of sitafloxacin were evaluated against clinical isolates of S. pneumoniae resistant to levofloxacin (MIC of levofloxacin > or = 4 mg/L), which were characterized genetically. METHODS: The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE of these strains were analysed by PCR-based sequencing. MICs of sitafloxacin and other quinolones were determined by a microdilution broth method. RESULTS: All 18 strains had at least one amino acid substitution in the QRDRs of GyrA and ParC, which included Ser-81-->Tyr/Phe and Glu-85-->Lys in GyrA and Ser-79-->Phe/Ile/Tyr, Asp-83-->Tyr, Asn-91-->Asp, Ser-107-->Phe, Lys-137-->Asn and Ala-142-->Ser in ParC. Most isolates had Asp-435-->Asn/Ile-460-->Val/Ala-596-->Thr substitutions in ParE, while no amino acid substitution in GyrB was noted in all isolates. Ten isolates for which levofloxacin MICs were 16 or 32 mg/L had multiple mutations in both GyrA and ParC. The MIC80 value of sitafloxacin for levofloxacin-resistant isolates was 0.25 mg/L. The range of MICs of sitafloxacin for isolates resistant to levofloxacin (MIC 4-32 mg/L) was 0.016-0.5 mg/L. CONCLUSIONS: These findings warrant further studies to evaluate the usefulness of sitafloxacin in the treatment of levofloxacin-resistant S. pneumoniae infection.     

Activities of clinafloxacin, gatifloxacin, gemifloxacin, and trovafloxacin against recent clinical isolates of levofloxacin-resistant Streptococcus pneumoniae

The activities of two investigational fluoroquinolones and three fluoroquinolones that are currently marketed were determined for 182 clinical isolates of Streptococcus pneumoniae. The collection included 57 pneumococcal isolates resistant to levofloxacin (MIC >/= 8 microg/ml) recovered from patients in North America and Europe. All isolates were tested with clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, and trovafloxacin by the National Committee for Clinical Laboratory Standards broth microdilution and disk diffusion susceptibility test methods. Gemifloxacin demonstrated the greatest activity on a per gram basis, followed by clinafloxacin, trovafloxacin, gatifloxacin, and levofloxacin. Scatterplots of the MICs and disk diffusion zone sizes revealed a well-defined separation of levofloxacin-resistant and -susceptible strains when the isolates were tested against clinafloxacin and gatifloxacin. DNA sequence analyses of the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE from 21 of the levofloxacin-resistant strains identified eight different patterns of amino acid changes. Mutations among the four loci had the least effect on the MICs of gemifloxacin and clinafloxacin, while the MICs of gatifloxacin and trovafloxacin increased by up to six doubling dilutions. These data indicate that the newer fluoroquinolones have greater activities than levofloxacin against pneumococci with mutations in the DNA gyrase or topoisomerase IV genes. Depending upon pharmacokinetics and safety, the greater potency of these agents could provide improved clinical efficacy against levofloxacin-resistant pneumococcal strains.     

南京地区肺炎链球菌的耐药变迁及喹诺酮耐药机制研究

目的了解南京地区肺炎链球菌临床分离株的耐药性变迁及对喹诺酮类的耐药机制。方法收集2010-2012年南京7所教学医院共147株肺炎链球菌,琼脂稀释法测定9种抗菌药物的MIC,与南京2006-2007年分离的肺炎链球菌耐药情况进行比较。并对氟喹诺酮耐药株的gyrA、gyrB、parE和parC基因喹喏酮耐药决定区域（QRDR）进行PCR扩增及测序。结果147株肺炎链球菌中,对红霉素的耐药率为89．1％;青霉素不敏感肺炎链球菌（MIC≥4mg／L）占3．4％;对头孢呋辛、头孢曲松、美罗培南、左氧氟沙星、莫西沙星的耐药率分别为26．5％、1．4％、3．4%、1．4％、0．7％;所有分离株对万古霉素、利奈唑胺敏感。与2006-2007年分离株相比,2010-2012年临床分离株对青霉素、头孢呋辛耐药率下降,对红霉素、头孢曲松耐药率变化不大,出现了少数左氧氟沙星及莫西沙星耐药株。对喹诺酮耐药株进行基因测序发现1菌株有gyrA突变（Asn167-Ile）和ParE突变（Ile460～Val）;另1菌株则有ParC突变（Ser81-Gly;Asn94-Asp）。结论南京地区肺炎链球菌对红霉素耐药率居高不下,对青霉素仍较敏感,出现了少数莫西沙星、左氧氟沙星耐药株。喹诺酮耐药株有gyrA、parE和parC的QRDR突变。     

Benchmarking the in vitro activities of moxifloxacin and comparator agents against recent respiratory isolates from 377 medical centers throughout the United States

To benchmark the activity of moxifloxacin (a newer fluoroquinolone), a U.S. study comprising 16,141 contemporary isolates of Streptococcus pneumoniae (5,640), Haemophilus influenzae (6,583), and Moraxella catarrhalis (3,648) referred from 377 institutions during 1998 is described. For S. pneumoniae the modal MIC and MIC at which 90% of the isolates were inhibited (MIC(90)) for moxifloxacin were 0.12 and 0.25 microg/ml, respectively, independent of susceptibility to other drug classes, geography, or site of infection. Eleven isolates were intermediate or resistant to levofloxacin and grepafloxacin; of these isolates, 1 remained susceptible to sparfloxacin, 2 remained susceptible to moxifloxacin, and 4 remained susceptible to trovafloxacin. All 11 isolates possessed classic mutations in gyrA and/or parC known to confer reduced susceptibility to fluoroquinolones. Four isolates (originating from four separate states) belonging to a multidrug-resistant, fluoroquinolone-resistant clone were identified by pulsed-field gel electrophoresis. For moxifloxacin and trovafloxacin, at least 87% of isolates demonstrated MICs > or =3 twofold concentrations below the susceptibility breakpoints, in contrast to no more than 15% for levofloxacin, grepafloxacin, and sparfloxacin. Of the isolates that were multidrug resistant (7.4%), >98% remained susceptible to moxifloxacin. The modal MIC and MIC(90) for M. catarrhalis (both 0.06 microg/ml) and for H. influenzae (both 0.03 microg/ml) were independent of beta-lactamase production. These data demonstrate the in vitro activity of moxifloxacin and establish a baseline for future studies.     

Comparative in vitro activity of telithromycin and beta-lactam antimicrobials against community-acquired bacterial respiratory tract pathogens in the United States: findings from the PROTEKT US study, 2000-2001

BACKGROUND: Telithromycin is a new ketolide antimicrobial that was developed to provide good activity against resistant respiratory tract pathogens. PROTEKT US (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin in the United States) is a multicenter in vitro surveillance study that was initiated in 2000 to chart the emergence and spread of antimicrobial-resistant pathogens in patients with community-acquired respiratory tract infections (CARTIs). OBJECTIVE: This article reports first-year results from PROTEKT US pertaining to the comparative in vitro activity of telithromycin and beta-lactam antimicrobials against community-acquired bacterial respiratory tract pathogens. METHODS: Data were compiled on the comparative in vitro activity of telithromycin and beta-lactams against Streptococcus pneumoniae (10,103 isolates), Streptococcus pyogenes (3918 isolates), and Haemophilus influenzae (2706 isolates). Minimum inhibitory concentrations (MICs) and susceptibilities were determined according to National Committee for Clinical Laboratory Standards methods. RESULTS: In total, 38.8% (3920/10,103) of pneumococcal isolates were not susceptible to penicillin (12.5% [1266] intermediate [MIC, > or =0.12-1.0 mg/dL], 26.3% [2654] resistant [MIC, > or =2 mg/dL]). Telithromycin was highly active against S pneumoniae (MIC required to inhibit 90% of isolates [MIC(90)], 0.5 mg/L), with 99.6% (10,062/10,103) of isolates fully susceptible (MIC, < or =1 mg/L). Based on MIC(90)s, the rank order of antimicrobial activity was telithromycin (0.5 mg/L), followed by amoxicillin/clavulanate (2 mg/L), penicillin (4 mg/L), and cefuroxime (8 mg/L). Telithromycin retained high activity (MIC(90), 1 mg/L) against penicillin-resistant penumococci that showed high levels of coresistance to beta-lactams. All isolates of S pyogenes were fully susceptible to the beta-lactams tested. Beta-lactamase production was common among H influenzae isolates (28.3% [765/2706]). Telithromycin was active against H influenzae (MIC(90), 4 mg/L), irrespective of beta-lactamase production. Conclusion: Overall, these findings from the first year of PROTEKT US support the potential value of telithromycin in the treatment of CARTIs.     

Fluoroquinolone resistance in Streptococcus pneumoniae in United States since 1994-1995

The in vitro activities of ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin against a large collection of clinical isolates of Streptococcus pneumoniae (n = 4,650) obtained over a 5-year period, 1994-1995 through 1999-2000, were assessed as part of a longitudinal multicenter U.S. surveillance study of antimicrobial resistance. Three sampling periods were used during this investigation, the winter seasons of 1994-1995, 1997-1998, and 1999-2000; and 1,523, 1,596 and 1,531 isolates were collected during these three periods, respectively. The overall rank order of activity of the four fluoroquinolones examined in this study was moxifloxacin > gatifloxacin > levofloxacin = ciprofloxacin, in which moxifloxacin (MIC at which 90% of isolates are inhibited [MIC(90)], 0.25 microg/ml; modal MIC, 0.12 microg/ml) was twofold more active than gatifloxacin (MIC(90), 0.5 microg/ml; modal MIC, 0.25 microg/ml), which in turn was fourfold more active than either levofloxacin (MIC(90), 1 microg/ml; modal MIC, 1 microg/ml) or ciprofloxacin (MIC(90), 2 microg/ml; modal MIC, 1 microg/ml). Changes in the in vitro activities of fluoroquinolones against S. pneumoniae strains in the United States over the 5-year period of the survey were assessed by comparing the MIC frequency distributions of the study drugs against the isolates obtained during the three sampling periods encompassing this investigation. These comparisons revealed no evidence of changes in the in vitro activities of the fluoroquinolones. In addition, the percentages of isolates in the three sampling periods for which MICs were above the resistance breakpoints were compared. Low percentages of resistant strains were detected, and there was no evidence of resistance rate changes over time. For example, by use of a ciprofloxacin MIC of > or = 4 microg/ml to define resistance, the proportions of isolates from the three sampling periods for which MICs were at or above this breakpoint were 1.2, 1.6, and 1.4%, respectively. A total of 164 unique isolates (n = 58 from 1994-1995, 65 from 1997-1998, and 42 from 1999-2000) were examined for evidence of mutations in the quinolone resistance-determining regions (QRDRs) of the parC and the gyrA genes. Forty-nine isolates harbored at least one mutation in the QRDRs of one or both genes (1994-1995, n = 15; 1997-1998, n = 19; 1999-2000, n = 15). Among the 4,650 isolates of S. pneumoniae examined in the study, we estimated that 0.3% had mutations in both the parC and gyrA loci. The majority of mutations (67.3% of the mutations in 49 isolates with mutations) were amino acid substitutions in the parC locus only. Four isolates had a mutation in the gyrA locus only, and 12 isolates had mutations in both genes (8.2 and 24.5% of isolates with mutations, respectively). There was no significant difference in the number of isolates with parC and/or gyrA mutations detected during each study period. Finally, because of the magnitude of the study, we had reasonably large numbers of pneumococcal isolates with genotypically defined mechanisms of fluoroquinolone resistance and were thus able to determine the effects of specific resistance mutations on the activities of different fluoroquinolones. In general, isolates with mutations in parC only were resistant to ciprofloxacin but remained susceptible to levofloxacin, gatifloxacin, and moxifloxacin, whereas isolates with mutations in gyrA only and isolates with mutations in both parC and gyrA were resistant to all four fluoroquinolones tested.