Safety, Reactogenicity, Immunogenicity, and Dose Selection of 10-Valent Extraintestinal Pathogenic Escherichia coli Bioconjugate Vaccine (VAC52416) in Adults Aged 60-85 Years in a Randomized, Multicenter, Interventional, First-in-Human, Phase 1/2a Study.

Background: ExPEC10V is a bioconjugate vaccine containing O-antigen polysaccharides of 10 extraintestinal pathogenic Escherichia coli (ExPEC) serotypes. This phase 1/2a study (NCT03819049) assessed the safety, reactogenicity, and immunogenicity of ExPEC10V (VAC52416) to prevent invasive E coli disease in elderly adults. Methods: The observer-blind, active-controlled design included a 28-day screening, vaccination, 181-day follow-up, and 1-year follow-up. Participants (60-85 years of age) were randomized to ExPEC10V low dose (antigen dose range, 4-8 µg), ExPEC10V medium dose (4-16 µg), or ExPEC10V high dose (8-16 µg); 4-valent ExPEC vaccine (ExPEC4V); or 13-valent pneumococcal conjugate vaccine (PCV13). The incidence of adverse events (AEs; solicited, day 15; unsolicited, day 30; serious AEs, day 181) and immunogenicity (electrochemiluminescent-based assay [ECL] and multiplex opsonophagocytic assay [MOPA]) were assessed. Optimal ExPEC10V dose was determined from safety data through day 30 and an immunogenicity dose selection algorithm based on day 15 ECL and MOPA results. Results: A total of 416 participants were included (median age, 64.0 years; 54.8% female). The incidences of solicited local and systemic AEs were, respectively, 44.2% and 39.4% for low-dose, 52.9% and 46.1% for medium-dose, 57.7% and 45.2% for high-dose ExPEC10V, and 74.1% and 48.1% for PCV13. Five serious AEs, not vaccine related, were reported. The ECL revealed a robust antibody response to ExPEC10V through year 1. Opsonophagocytic killing activity was detected against all but serotype O8; this lack of response against serotype O8 was linked to low assay sensitivity. Based on the totality of data, high-dose ExPEC10V was considered optimal. Conclusions: ExPEC10V was well tolerated and immunogenic in elderly adults against all but serotype O8.

Development and Qualification of an Opsonophagocytic Killing Assay To Assess Immunogenicity of a Bioconjugated Escherichia coli Vaccine.

The global burden of disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) is increasing as the prevalence of multidrug-resistant strains rises. A multivalent ExPEC O-antigen bioconjugate vaccine could have a substantial impact in preventing bacteremia and urinary tract infections. Development of an ExPEC vaccine requires a readout to assess the functionality of antibodies. We developed an opsonophagocytic killing assay (OPA) for four ExPEC serotypes (serotypes O1A, O2, O6A, and O25B) based on methods established for pneumococcal conjugate vaccines. The performance of the assay was assessed with human serum by computing the precision, linearity, trueness, total error, working range, and specificity. Serotypes O1A and O6A met the acceptance criteria for precision (coefficient of variation for repeatability and intermediate precision, ≤50%), linearity (90% confidence interval of the slope of each strain, 0.80, 1.25), trueness (relative bias range, -30% to 30%), and total error (total error range, -65% to 183%) at five serum concentrations and serotypes O2 and O25B met the acceptance criteria at four concentrations (the lowest concentration for serotypes O2 and O25B did not meet the system suitability test of maximum killing of ≥85% of E. coli cells). All serotypes met the acceptance criteria for specificity (opsonization index value reductions of ≤20% for heterologous serum preadsorption and ≥70% for homologous serum preadsorption). The assay working range was defined on the basis of the lowest and highest concentrations at which the assay jointly fulfilled the target acceptance criteria for linearity, precision, and accuracy. An OPA suitable for multiple E. coli serotypes has been developed, qualified, and used to assess the immunogenicity of a 4-valent E. coli bioconjugate vaccine (ExPEC4V) administered to humans.

Evaluation of clonality and carbapenem resistance mechanisms among Acinetobacter baumannii-Acinetobacter calcoaceticus complex and Enterobacteriaceae isolates collected in European and Mediterranean countries and detection of two novel ß-lactamases, GES-22 and VIM-35.

We evaluated doripenem-resistant Acinetobacter baumannii-Acinetobacter calcoaceticus complex (ACB; n = 411) and Enterobacteriaceae (n = 92) isolates collected from patients from 14 European and Mediterranean countries during 2009 to 2011 for the presence of carbapenemase-encoding genes and clonality. Following susceptibility testing, carbapenem-resistant (doripenem MIC, >2 µg/ml) isolates were screened for carbapenemases. New ß-lactamase genes were expressed in a common background and susceptibility was tested. Class 1 integrons were sequenced. Clonality was evaluated by pulsed-field gel electrophoresis and multilocus sequence typing (Pasteur scheme). Relative expression of ß-lactam intrinsic resistance mechanisms was determined for carbapenemase-negative Enterobacteriaceae. ACB and Enterobacteriaceae displayed 58.9 and 0.9% doripenem resistance, respectively. bla(OXA-23), bla(OXA-58), and bla(OXA-24/OXA-40) were detected among 277, 77, and 29 ACB, respectively (in 8, 6, and 5 countries). Ten Turkish isolates carried bla(GES-11) or bla(GES-22). GES-22 (G243A and M169L mutations in GES-1) had an extended-spectrum ß-lactamase profile. A total of 33 clusters of ≥ 2 ACB isolates were observed, and 227 isolates belonged to sequence type 2/international clone II. Other international clones were limited to Turkey and Israel. Doripenem-resistant Enterobacteriaceae increased significantly (0.7 to 1.6%), and 15 blaKPC-2- and 22 blaKPC-3-carrying isolates, mostly belonging to clonal complexes 11 and 258, were observed. Enterobacteriaceae isolates producing OXA-48 (n = 16; in Turkey and Italy), VIM-1 (n = 10; in Greece, Poland, and Spain), VIM-26 (n = 1; in Greece), and IMP-19, VIM-4, and the novel VIM-35 (n = 1 each from Poland) were detected. VIM-35 had one substitution compared to VIM-1 (A235T) and a similar susceptibility profile. One or more resistance mechanisms were identified in 4/6 carbapenemase-negative Enterobacteriaceae. This broad evaluation confirms results from country-specific surveys and shows a highly diverse population of carbapenemase-producing ACB and Enterobacteriaceae in Europe and Mediterranean countries.

Epidemiology and carbapenem resistance mechanisms of carbapenem-non-susceptible Pseudomonas aeruginosa collected during 2009-11 in 14 European and Mediterranean countries.

OBJECTIVES: To evaluate the genetic relatedness and carbapenem resistance mechanisms among carbapenem-non-susceptible Pseudomonas aeruginosa collected during 2009-11 in 14 European and Mediterranean countries. METHODS: Doripenem-non-susceptible (MIC >2 mg/L) isolates were tested for susceptibility to imipenem, meropenem, doripenem, aztreonam, ceftazidime and cefepime with and without phenyl-arginine-ß-naphthylamide (PAßN) (efflux inhibitor) and/or cloxacillin (AmpC inhibitor). Carbapenemase screening was performed by PCR and sequencing. Expression of chromosomal ampC, mexA, mexC, mexE and mexX was determined by quantitative real-time PCR using P. aeruginosa PAO1 or a group of susceptible isolates as baseline. Clonality was evaluated by PFGE and multilocus sequence typing. RESULTS: Among 529 (25.6% overall) carbapenem-non-susceptible P. aeruginosa, 106 were positive for metallo-ß-lactamase (MßL) genes encoding VIM-2 (76 strains), VIM-4 (14), VIM-1 (7) and VIM-5 (5). IMP-15 and three new MßLs (IMP-33, VIM-36 and VIM-37) were detected in one strain each. An increasing prevalence of MßL producers was noted in 2011 (30.6%) compared with previous years (13.4% and 12.3% in 2009 and 2010, respectively). Isolates displayed high genetic diversity, with 401 unique profiles detected. CC235 and ST111 were detected among MßL-producing clusters. The PAßN/cloxacillin effect ranged from 90.0% to 56.5%/from 1.3% to 21.2%. OprD decrease/loss was the most prevalent intrinsic mechanism and was detected among 94.9% of the P. aeruginosa, followed by AmpC (44.4%) and MexAB-OprM (20.1%). When using the susceptible group of isolates as baseline, MexAB-OprM became as prevalent as OprD decrease/loss. CONCLUSIONS: Increasing MßL prevalence is worrisome in various European countries; however, intrinsic resistance mechanisms in a highly genetically diverse population of carbapenem-non-susceptible P. aeruginosa are probably a matter for greater concern in these countries.

Assessment of the combination of doripenem plus a fluoroquinolone against non-susceptible Acinetobacter baumannii isolates from nosocomial pneumonia patients.

Carbapenem- and fluoroquinolone-non-susceptible Acinetobacter baumannii were obtained from four nosocomial pneumonia patients who were clinically cured following combination therapy with doripenem/levofloxacin or ciprofloxacin. In vitro synergy of doripenem/levofloxacin or ciprofloxacin was evaluated using time-kill analysis. In vivo synergy was tested using a mouse lethal infection model. In time-kill studies, doripenem and levofloxacin were both bactericidal when tested at Cmax; at ½Cmax, the combination showed synergy up to 8 hours. Ciprofloxacin, alone or combined with doripenem, was not bactericidal. For mouse septicemia, doripenem (100 mg/kg) was ≥90% effective in preventing death in all four isolates. Levofloxacin (200 mg/kg) was 73% effective, and ciprofloxacin (35 mg/kg) was ineffective in preventing death. At lower drug concentrations, increased efficacy was observed for doripenem/levofloxacin, but not for doripenem/ciprofloxacin. Overall, the results suggest that a doripenem/levofloxacin combination may have clinical utility in treating some non-susceptible A. baumannii infections.

Activities of carbapenem and comparator agents against contemporary US Pseudomonas aeruginosa isolates from the CAPITAL surveillance program.

Antimicrobial susceptibilities of contemporary Pseudomonas aeruginosa clinical isolates were determined from the CAPITAL 2010 surveillance program. Isolates were collected from 100 sites throughout the USA and Puerto Rico, and included isolates representing a range of patient demographics and infection types. A total of 2722 isolates were tested for susceptibility to a broad spectrum of agents, with susceptibilities ranging from 98.8% for colistin to 74% for levofloxacin. Doripenem was the most active carbapenem agent, with 88.6% of isolates susceptible, in comparison with 78.1% and 84.6% for imipenem and meropenem, respectively. Lower respiratory tract isolates and isolates from the intensive care unit setting were the least susceptible overall. Resistance rates were typically highest in lower respiratory tract isolates, with the exception of urinary tract isolates, which displayed the highest resistance for levofloxacin. Overall, multidrug-resistant isolates comprised 14.8% of the total sample population.

In vitro Etest synergy of doripenem with amikacin, colistin, and levofloxacin against Pseudomonas aeruginosa with defined carbapenem resistance mechanisms as determined by the Etest method.

The applicability of Etest for synergy testing was evaluated in 100 carbapenem-nonsusceptible Pseudomonas aeruginosa isolates. Combinations of doripenem with amikacin, colistin, or levofloxacin were synergistic or additive against 67%, 31%, and 23% of isolates, respectively. The use of Etest was practical to evaluate the synergy of doripenem with other antipseudomonal agents.

Multidrug resistance among Acinetobacter spp. in the USA and activity profile of key agents: results from CAPITAL Surveillance 2010.

Multidrug resistance among Acinetobacter spp. leaves few effective antibiotic options for treatment. To monitor antibiotic resistance in Acinetobacter spp., the US CAPITAL 2010 Surveillance data were evaluated by patient demographics, specimen source, and hospital ward. Isolates (N=514) were collected from 65 sites across the USA and Puerto Rico. Isolates were centrally tested for susceptibility to carbapenems and key antimicrobials by broth microdilution. Colistin was the most effective agent tested, with 95% susceptibility. The overall susceptibility of Acinetobacter spp. was low (39% for piperacillin/tazobactam, 41% for levofloxacin, 45% for ceftazidime, 47-51% for the carbapenems, and 58% for tobramycin). Multidrug resistance (MDR), defined as resistance to ≥3 antimicrobial agent groups, was detected in 54% of the isolates. MDR isolates were most common among elderly patients (65%), lower respiratory tract isolates (62%), and inpatient/intensive care unit isolates (54-58%). These data update trends in the distribution and prevalence of the MDR phenotype in Acinetobacter spp.

Activity of ceftobiprole against Streptococcus pneumoniae isolates exhibiting high-level resistance to ceftriaxone.

Tracking Resistance in the US Today (TRUST) 2008 surveillance data showed that 6% of Streptococcus pneumoniae were non-susceptible to ceftriaxone [minimum inhibitory concentration (MIC) ≥ 2 µg/mL] and that 8% of the ceftriaxone-non-susceptible isolates exhibited high-level resistance (MIC ≥ 8 µg/mL). Here we describe the activity of ceftobiprole against ceftriaxone-resistant isolates and characterise the genotypic traits associated with resistance. Thirty isolates with ceftriaxone MICs ≥ 8 µg/mL were analysed by sequencing of penicillin-binding protein (PBP) and murM genes. Sequencing of pbp1a, pbp2b and pbp2x showed nine PBP patterns, with the most common (n=17) being: PBP1a T371S (STMK motif), P432T (SRNVP motif); PBP2b T446A (SSNT motif), A619G (KTGTA motif); and PBP2x T338A and M339F (STMK motif), L364F, I371T, R384G, M400T, L546V (LKSGT motif); six isolates had the same pattern without the PBP2b A619G change. For these 23 isolates, MICs were 8 µg/mL for ceftriaxone, 4-8 µg/mL for penicillin and 0.5-2 µg/mL for ceftobiprole. The remaining seven isolates with higher MICs (ceftriaxone 8-32 µg/mL, penicillin 4-32 µg/mL and ceftobiprole 2-4 µg/mL) had fewer PBP active-site motif substitutions. The majority of isolates (17/30) had murM alleles similar to the wild-type, whilst the rest had alleles reflecting a mosaic structure. No murM alleles were associated with higher MICs. Against these 30 isolates, ceftobiprole was 4-16-fold more active than ceftriaxone. Widely described PBP and MurM substitutions probably account for the high ceftriaxone MICs (8 µg/mL) in the majority of isolates. However, seven isolates with ceftriaxone MICs of 8-32 µg/mL had fewer PBP substitutions in active-site motifs, suggesting either that there is another resistance mechanism or that unique PBP mutations may contribute to high-level ß-lactam resistance.

Antistaphylococcal activities of the new fluoroquinolone JNJ-Q2.

The new broad-spectrum fluoroquinolone JNJ-Q2 displays in vitro activity against Gram-negative and Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant MRSA isolates. Tested with isogenic methicillin-susceptible S. aureus (MSSA) and MRSA strains bearing quinolone-resistant target mutations, JNJ-Q2 displayed MICs ≤ 0.12 µg/ml, values 16- to 32-fold lower than those determined for moxifloxacin. Overexpression of the NorA efflux pump did not impact JNJ-Q2 MICs. Inhibition of S. aureus DNA gyrase and DNA topoisomerase IV enzymes demonstrated that JNJ-Q2 was more potent than comparators against wild-type enzymes and enzymes carrying quinolone-resistant amino acid substitutions, and JNJ-Q2 displayed equipotent activity against both enzymes. In serial-passage studies comparing resistance selection in parallel MRSA cultures by ciprofloxacin and JNJ-Q2, ciprofloxacin readily selected for mutants displaying MIC values of 128 to 512 µg/ml, which were observed within 18 to 24 days of passage. In contrast, cultures passaged in the presence of JNJ-Q2 displayed MICs ≤ 1 µg/ml for a minimum of 27 days of serial passage. A mutant displaying a JNJ-Q2 MIC of 4 µg/ml was not observed until after 33 days of passage. Mutant characterization revealed that ciprofloxacin-passaged cultures with MICs of 256 to 512 µg/ml carried only 2 or 3 quinolone resistance-determining region (QRDR) mutations. Cultures passaged with JNJ-Q2 selection for up to 51 days displayed MICs of 1 to 64 µg/ml and carried between 4 and 9 target mutations. Established in vitro biofilms of wild-type or ciprofloxacin-resistant MRSA exposed to JNJ-Q2 displayed greater decreases in bacterial counts (7 days of exposure produced 4.5 to >7 log(10) CFU decreases) than biofilms exposed to ciprofloxacin, moxifloxacin, rifampin, or vancomycin.

Longitudinal survey of carbapenem resistance and resistance mechanisms in Enterobacteriaceae and non-fermenters from the USA in 2007-09.

BACKGROUND: Antibiotic resistance is problematic in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, and is often associated with serious infections. Carbapenems are often one of the few remaining therapeutic options, so it is important to monitor carbapenem activity against these pathogens and to identify resistance mechanisms. METHODS: Carbapenem susceptibilities were determined for 14 359 Enterobacteriaceae, 3614 P. aeruginosa and 994 A. baumannii from the USA (2007-09). Klebsiella pneumoniae with doripenem MICs ≥2 mg/L (n = 88), and P. aeruginosa (n = 452), A. baumannii (n = 349) and other enterics (n = 13) with doripenem MICs ≥4 mg/L were screened for carbapenem resistance mechanisms. RESULTS: Doripenem/meropenem and imipenem susceptibilities for Enterobacteriaceae were >99% and 89%, respectively. Doripenem susceptibility (2007-09) for P. aeruginosa was 87.4%-84.1%; comparable to meropenem and higher than imipenem. For A. baumannii, doripenem susceptibility (2007-09) was 63%-58.2%; lower than imipenem and meropenem. Resistant K. pneumoniae had KPC and lacked porins OmpK35/OmpK36. In 2009, 3.4% of all K. pneumoniae possessed KPC. Five other enterics and one P. aeruginosa possessed KPC. Resistance mechanisms in P. aeruginosa were loss of porin OprD (90%), efflux (55%) and elevated AmpC activity (25%). Acquired carbapenemases OXA-23/-24 were present in 48% of resistant A. baumannii. VIM metallo-ß-lactamases were present in three P. aeruginosa and one A. baumannii isolates. CONCLUSIONS: Doripenem and meropenem were more active than imipenem against Enterobacteriaceae and P. aeruginosa from the USA. Carbapenem resistance mechanisms included serine carbapenemases, elevated AmpC activity, efflux and porin deficiencies occurring mostly in P. aeruginosa. Metallo-ß-lactamases were found in <0.1% of isolates.

Affinity of ceftobiprole for penicillin-binding protein 2b in Streptococcus pneumoniae strains with various susceptibilities to penicillin.

Wild-type penicillin-binding protein (PBP) 2b from penicillin-susceptible Streptococcus pneumoniae had high affinity for ceftobiprole and penicillin (50% inhibitory concentrations [IC(50)s] of ≤0.15 µg/ml) but not ceftriaxone (IC(50) of >8 µg/ml). In clinical isolates, ceftobiprole and PBP 2b affinities were reduced 15- to 30-fold with a Thr-446-Ala substitution and further still with an additional Ala-619-Gly PBP 2b substitution. Ceftobiprole remained active (MICs of ≤1 µg/ml) against all strains tested and behaved more like penicillin than ceftriaxone with respect to PBP 2b binding.

Effects of ceftobiprole and oxacillin on mecA expression in methicillin-resistant Staphylococcus aureus clinical isolates.

Induction of mecA by ceftobiprole and oxacillin in 18 methicillin-resistant Staphylococcus aureus clinical isolates with various SCCmec cassettes was examined using reverse transcriptase PCR. The magnitude of mecA induction, 3- to 65-fold for ceftobiprole and 2- to 69-fold for oxacillin, did not correlate with ceftobiprole MICs (or=256 microg/ml. No correlation between magnitude of induction and SCCmec type was found.

Effect of MexXY overexpression on ceftobiprole susceptibility in Pseudomonas aeruginosa.

Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus broad-spectrum cephalosporin, has activity (MIC for 50% of strains tested, < or =4 microg/ml) against many Pseudomonas aeruginosa strains. A common mechanism of P. aeruginosa resistance to beta-lactams, including cefepime and ceftazidime, is efflux via increased expression of Mex pumps, especially MexAB. MexXY has differential substrate specificity, recognizing cefepime but not ceftazidime. In ceftobiprole clinical studies, paired isolates of P. aeruginosa from four subjects demonstrated ceftobiprole MICs of 2 to 4 microg/ml at baseline but 16 microg/ml posttreatment, unrelated to beta-lactamase levels. Within each pair, the level of mexXY RNA, but not mexAB, mexCD, and mexEF, increased by an average of 50-fold from baseline to posttreatment isolates. Sequencing of the negative regulatory gene mexZ indicated that each posttreatment isolate contained a mutation not present at baseline. mexXY expression as a primary ceftobiprole and cefepime resistance mechanism was further examined in isogenic pairs by using cloned mexXY and mexZ. Expression of cloned mexXY in strain PAO1 or in a baseline isolate increased the ceftobiprole MIC to that for the posttreatment isolate. In contrast, in posttreatment isolates, lowering mexXY expression via introduction of cloned mexZ decreased the ceftobiprole MIC to that for the baseline isolates. Similar changes were observed for cefepime. A spontaneous mutant selectively overexpressing mexXY displayed a fourfold elevation in its ceftobiprole MIC, while overexpression of mexAB, -CD, and -EF had a minimal effect. These data indicate that ceftobiprole, like cefepime, is an atypical beta-lactam that is a substrate for the MexXY efflux pump in P. aeruginosa.

Molecular characterisation of meticillin-resistant Staphylococcus aureus isolates from two ceftobiprole Phase 3 complicated skin and skin-structure infection clinical trials.

Meticillin-resistant Staphylococcus aureus (MRSA) isolates from two worldwide ceftobiprole Phase 3 clinical trials for the treatment of complicated skin and skin-structure infections were characterised by clonality, staphylococcal cassette chromosome mec (SCCmec) type and the presence of Panton-Valentine leukocidin (PVL). PVL was predominantly found in US isolates (196/231 vs. 13/110 non-US isolates). SCCmec type IV was the most common (253/329) owing to the predominance of clone USA300 in isolates from the USA (197/226). In Europe, SCCmec type III was the most prevalent (30/74). Ceftobiprole minimum inhibitory concentrations (MICs) ranged from 0.25 microg/mL to 4 microg/mL, with MICs

Sporadic occurrences of fluoroquinolone-resistant Streptococcus pneumoniae in the United States: longitudinal analysis of institutions from the New England and West South Central regions during the TRUST 4-9 (2000-2005) Surveillance Studies.

Twenty-six institutions in New England and 24 institutions in West South Central regions participating in the Tracking Resistance in the United States Today (TRUST) 4-9 surveillance studies (2000-2005) were monitored for levofloxacin-resistant Streptococcus pneumoniae to determine if resistance was sporadic or persistent. Levofloxacin was used as a representative of the respiratory fluoroquinolones. Levofloxacin-resistant isolates were identified in 8 of the 26 New England institutions and in 11 of the 24 West South Central institutions during the surveillance period. Resistant isolates were recovered in consecutive years from 3 institutions: 1 each in Massachusetts, Oklahoma, and Texas. In total, 34 levofloxacin-resistant isolates (14 from New England, 20 from the West South Central region) were identified over the 6-year period. Two of these isolates from an institution in Connecticut and 2 from an institution in Oklahoma had the same serotype, pulsed-field gel electrophoresis pattern, and quinolone resistance-determining region (QRDR) mutations. States with elevated pneumococcal levofloxacin resistance rates, compared with the national average, did not maintain this status in consecutive years. Based on data from the same institutions over 6 years, levofloxacin resistance among US pneumococci has been sporadic, nonclonal, and rare.

Decline in the prevalence of pandemic clones Spain23F-1 and Spain9V-3 among US fluoroquinolone-resistant Streptococcus pneumoniae TRUST Surveillance isolates since 2001.

Fluoroquinolone-resistant variants of pandemic clones Spain(23F)-1, Spain(6B)-2, Spain(9V)-3, and Spain(14)-5 have been seen in various regions of the United States and the world, leading to the speculation that fluoroquinolone resistance among US Streptococcus pneumoniae may increase because of clonal spread. Using levofloxacin as a representative of the fluoroquinolone class, all 196 levofloxacin-resistant pneumococci from a total of 22 794 isolates in the Tracking Resistance in the United States Today (TRUST) 5-8 studies (2001-2004) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping, and sequencing of parC/E and gyrA/B quinolone resistance-determining regions (QRDR) to measure the extent of clonality. In addition, susceptibility testing of these isolates to ciprofloxacin, gatifloxacin, levofloxacin, and moxifloxacin was performed. ATCC type strains of Spain(23F)-1, Spain(6B)-2, Spain(9V)-3, and Spain(14)-5 clones were included as comparators. Levofloxacin-resistant isolates with Spain(23F)-1-related PFGE patterns decreased from 28% of the resistant isolates in 2001 to 6% in 2004. These isolates, with serotypes 23F (n = 17), 19F (n = 17), or 19A (n = 1), had 15 different QRDR profiles and were all ciprofloxacin- and gatifloxacin-resistant. Levofloxacin-resistant isolates with Spain(9V)-3-related PFGE patterns decreased from 13% of the resistant isolates in 2001 to 2% in 2004. The Spain(9V)-3-related isolates were serotype 9V (n = 9), 9A (n = 2), and 9N (n = 1) and exhibited 6 different QRDR profiles. All were resistant to all fluoroquinolones tested. None of the levofloxacin-resistant isolates had PFGE patterns related to Spain(6B)-2 or Spain(14)-5. Resistance to respiratory fluoroquinolones among pneumococci has remained constant at about 1% (0.8%-1.1%) since 2001, and there has been a decrease in the prevalence of levofloxacin-resistant isolates similar to Spain(23F)-1 or Spain(9V)-3. Considerable QRDR variability among these strains appears to be the result of sporadic independent mutational events as opposed to clonal expansion.

Effects of the 7-valent pneumococcal conjugate vaccine on U.S. levofloxacin-resistant Streptococcus pneumoniae.

BACKGROUND: Seven-valent pneumococcal conjugate vaccine (PCV7) provides protection against invasive pneumococcal disease that extends to unvaccinated populations, such as elderly or immunocompromised adults. PCV7 also reduces incidence of pneumococcal penicillin resistance. In this study, the potential impact of PCV7 on pneumococcal fluoroquinolone resistance was examined. METHODS: U.S. levofloxacin-resistant isolates (264) from TRUST surveillance studies (1999-2004) were serotyped and quinolone resistance-determining region of parC/E and gyrA/B sequenced. Changes in prevalence of vaccine/nonvaccine serotypes during 2000-2004 and 1999-2004 were analyzed by regression analyses and chi-square trend test. RESULTS: The introduction of PCV7 (2000-2004) did not affect fluoroquinolone resistance prevalence, but mutants with vaccine serotypes declined linearly at -6.6 +/- 0.8% per year (p = 0.003), with concomitant replacement by nonvaccine serotypes; vaccine-related serotypes (6A, 9N, 19A, and 23N) increased (p = 0.04). Differential selection between vaccine and nonvaccine serotypes occurred for mutants containing amino acid substitutions at either ParC Ser79 (p = 0.01) or both ParC Ser79 and GyrA Ser81 (p = 0.04). Among mutants with ParC Ser79 substitutions, vaccine serotypes declined linearly (p = 0.02), whereas nonvaccine serotypes increased (p = 0.04). Additionally, a vaccine-independent effect became apparent during 1999-2004, as the incidence of ParC Ser79 and Asp83 mutations declined in fluoroquinolone-resistant strains, suggesting that these substitutions conferred decreased fitness. CONCLUSIONS: PCV7 has led to extensive replacement of vaccine serotypes by nonvaccine serotypes among levofloxacin-resistant pneumococci.

In vitro activity of ceftobiprole against pathogens from two phase 3 clinical trials of complicated skin and skin structure infections.

In phase 3 clinical trials for ceftobiprole treatment of complicated skin and skin structure infections, 1,219 gram-positive and 276 gram-negative aerobic baseline pathogens were identified. Ceftobiprole inhibited all staphylococcal isolates, including methicillin-resistant strains, at MICs of