Signal Biosynthesis Inhibition with Ambuic Acid as a Strategy To Target Antibiotic-Resistant Infections.

There has been major interest by the scientific community in antivirulence approaches against bacterial infections. However, partly due to a lack of viable lead compounds, antivirulence therapeutics have yet to reach the clinic. Here we investigate the development of an antivirulence lead targeting quorum sensing signal biosynthesis, a process that is conserved in Gram-positive bacterial pathogens. Some preliminary studies suggest that the small molecule ambuic acid is a signal biosynthesis inhibitor. To confirm this, we constructed a methicillin-resistant Staphylococcus aureus (MRSA) strain that decouples autoinducing peptide (AIP) production from regulation and demonstrate that AIP production is inhibited in this mutant. Quantitative mass spectrometric measurements show that ambuic acid inhibits signal biosynthesis (50% inhibitory concentration [IC50] of 2.5 ± 0.1 µM) against a clinically relevant USA300 MRSA strain. Quantitative real-time PCR confirms that this compound selectively targets the quorum sensing regulon. We show that a 5-µg dose of ambuic acid reduces MRSA-induced abscess formation in a mouse model and verify its quorum sensing inhibitory activity in vivo Finally, we employed mass spectrometry to identify or confirm the structure of quorum sensing signaling peptides in three strains each of S. aureus and Staphylococcus epidermidis and single strains of Enterococcus faecalis, Listeria monocytogenes, Staphylococcus saprophyticus, and Staphylococcus lugdunensis By measuring AIP production by these strains, we show that ambuic acid possesses broad-spectrum efficacy against multiple Gram-positive bacterial pathogens but does not inhibit quorum sensing in some commensal bacteria. Collectively, these findings demonstrate the promise of ambuic acid as a lead for the development of antivirulence therapeutics.

Changes in pneumococcal serotypes and antimicrobial resistance after introduction of the 13-valent conjugate vaccine in the United States.

Ongoing surveillance for Streptococcus pneumoniae is needed to assess the impact of the pneumococcal conjugate vaccine introduced in 2010 (PCV13). Forty-two U.S. centers submitted S. pneumoniae isolates between 1 October 2012 and 31 March 2013. Susceptibility testing was performed by use of a broth dilution method as recommended by the Clinical and Laboratory Standards Institute. Serotyping was performed by multiplex PCR and the Quellung reaction. Multidrug resistance (MDR) was defined as nonsusceptibility to penicillin (PNSP; MIC ≥ 0.12 µg/ml) combined with resistance to ≥2 non-ß-lactam antimicrobials. Penicillin-resistant S. pneumoniae (PRSP) was defined as a penicillin MIC of ≥2 µg/ml. For the 1,498 isolates collected during 2012-13, the PRSP and MDR rates were 14.2 and 21.0%, respectively. These percentages were lower than rates obtained in a surveillance study conducted 4 years earlier in 2008-09 (17.0 and 26.6%, respectively). The most common serotypes identified in 2012-13 were 3, 35B, and 19A, each representing 9 to 10% of all isolates. The largest percentage of PNSP in 2012-13 were found in serotypes 35B (24.8%), 19A (23.5%), and 15A (10.3%). Predominant PRSP serotypes were 19A (54.5%), 35B (28.2%), and 19F (7.0%). Major MDR serotypes were 19A (38.5%), 15A (16.9%), 6C (8.3%), and 35B (6.4%). The change in prevalence of PCV13 serotypes (43.4 to 27.1%) was primarily due to a decrease in serotype 19A strains, i.e., 22% of all strains in 2008-09 to 10% of all strains in 2012-13. Among the PNSP subset, serotypes showing a proportional increase were 35B, 15B, and 23B. Among MDR strains, the largest proportional increases were observed in serotypes 35B, 15B, and 23A.

Activities of vancomycin, ceftaroline, and mupirocin against Staphylococcus aureus isolates collected in a 2011 national surveillance study in the United States.

Forty-two medical centers from throughout the United States participating in a longitudinal surveillance program were asked to submit 100 consecutive Staphylococcus aureus isolates during July to December 2011. Susceptibility testing using CLSI broth microdilution and mecA detection by PCR analysis was performed on the 4,131 isolates collected. Methods employing Etest glycopeptide resistance detection (GRD; bioMérieux) and brain heart infusion agar containing 4 µg/ml vancomycin (BHIV) were used to screen methicillin-resistant S. aureus (MRSA) isolates for heterogeneous intermediate-level resistance to vancomycin (hVISA). Isolates with positive hVISA screen results were confirmed by population analysis profiling-area under the curve (PAP-AUC) determinations. The genetic relatedness of hVISA, ceftaroline-nonsusceptible, or high-level (HL) mupirocin resistance MRSA isolates was assessed by pulsed-field gel electrophoresis (PFGE). Among 2,093 MRSA isolates, the hVISA screen results were positive with 47 isolates by Etest GRD and 30 isolates by BHIV agar screen. Twenty-five of the GRD- or BHIV screen-positive isolates were confirmed as hVISA by PAP-AUC testing. Results of the current study were compared to results obtained from prior surveillance performed in 2009. The prevalence of hVISA among MRSA isolates was higher in 2011 than in 2009 (1.2% versus 0.4%, P = 0.003), especially for isolates with a vancomycin MIC of 2 (45.4% versus 14.3%, P = 0.01). The overall rate of ceftaroline susceptibility in the current study was 99.4% (one hVISA isolate had an intermediate ceftaroline MIC). HL mupirocin resistance increased from 2.2% in 2009 to 3.2% in 2011 (P = 0.006). Although overall rates of hVISA and HL mupirocin resistance are low, they have increased since 2009.

Pneumococcal serotypes before and after introduction of conjugate vaccines, United States, 1999-2011(1.).

Serotyping data for pneumococci causing invasive and noninvasive disease in 2008-2009 and 2010-2011 from >43 US centers were compared with data from preconjugate vaccine (1999-2000) and postconjugate vaccine (2004-2005) periods. Prevalence of 7-valent pneumococcal conjugate vaccine serotypes decreased from 64% of invasive and 50% of noninvasive isolates in 1999-2000 to 3.8% and 4.2%, respectively, in 2010-2011. Increases in serotype 19A stopped after introduction of 13-valent pneumococcal vaccine (PCV13) in 2010. Prevalences of other predominant serotypes included in or related to PCV13 (3, 6C, 7F) also remained similar for 2008-2009 and 2010-2011. The only major serotype that increased from 2008-2009 to 2010-2011 was nonvaccine serotype 35B. These data show that introduction of the 7-valent vaccine has dramatically decreased prevalence of its serotypes and that addition of serotypes in PCV13 could provide coverage of 39% of isolates that continue to cause disease.

Evaluation of pneumococcal serotyping by multiplex PCR and quellung reactions.

Screening of 1,750 pneumococcal isolates for common serotypes by PCR was followed by Quellung reaction analysis of PCR-negative isolates with a comparison to the conventional (Quellung reaction only) approach. PCR agreed with Quellung reaction results for 99% of isolates. The sequential PCR/Quellung reaction algorithm is accurate and more cost-effective than the conventional approach.

In vitro activity of ceftaroline against clinical isolates of Streptococcus pneumoniae recovered in 43 U.S. medical centers during 2010-2011.

The in vitro activity of ceftaroline, a recently introduced parenteral cephalosporin, was assessed versus 1,750 isolates of Streptococcus pneumoniae recovered from patients with a variety of pneumococcal infections in 43 U.S. medical centers during 2010-2011. Using a breakpoint of ≤ 0.5 µg/ml for susceptibility, all of the isolates were found to be susceptible to ceftaroline. Ceftaroline MICs were consistently 16-fold lower than ceftriaxone MICs. Among the isolates characterized in this investigation, 38.9% were found to be nonsusceptible to penicillin (oral penicillin breakpoints) and 9.1% were nonsusceptible to ceftriaxone (nonmeningitis breakpoints).

Activity of ceftaroline and epidemiologic trends in Staphylococcus aureus isolates collected from 43 medical centers in the United States in 2009.

A Staphylococcus aureus surveillance program was initiated in the United States to examine the in vitro activity of ceftaroline and epidemiologic trends. Susceptibility testing by Clinical and Laboratory Standards Institute broth microdilution was performed on 4,210 clinically significant isolates collected in 2009 from 43 medical centers. All isolates were screened for mecA by PCR and evaluated by pulsed-field gel electrophoresis. Methicillin-resistant S. aureus (MRSA) were analyzed for Panton-Valentine leukocidin (PVL) genes and the staphylococcal cassette chromosome mec (SCCmec) type. All isolates had ceftaroline MICs of ≤2 µg/ml with an MIC(50) of 0.5 and an MIC(90) of 1 µg/ml. The overall resistance rates, expressed as the percentages of isolates that were intermediate and resistant (or nonsusceptible), were as follows: ceftaroline, 1.0%; clindamycin, 30.2% (17.4% MIC ≥ 4 µg/ml; 12.8% inducible); daptomycin, 0.2%; erythromycin, 65.5%; levofloxacin, 39.9%; linezolid, 0.02%; oxacillin, 53.4%; tetracycline, 4.4%; tigecycline, 0%; trimethoprim-sulfamethoxazole, 1.6%; vancomycin, 0%; and high-level mupirocin, 2.2%. The mecA PCR was positive for 53.4% of the isolates. The ceftaroline MIC(90)s were 0.25 µg/ml for methicillin-susceptible S. aureus and 1 µg/ml for MRSA. Among the 2,247 MRSA isolates, 51% were USA300 (96.9% PVL positive, 99.7% SCCmec type IV) and 17% were USA100 (93.4% SCCmec type II). The resistance rates for the 1,137 USA300 MRSA isolates were as follows: erythromycin, 90.9%; levofloxacin, 49.1%; clindamycin, 7.6% (6.2% MIC ≥ 4 µg/ml; 1.4% inducible); tetracycline, 3.3%; trimethoprim-sulfamethoxazole, 0.8%; high-level mupirocin, 2.7%; daptomycin, 0.4%; and ceftaroline and linezolid, 0%. USA300 is the dominant clone causing MRSA infections in the United States. Ceftaroline demonstrated potent in vitro activity against recent S. aureus clinical isolates, including MRSA, daptomycin-nonsusceptible, and linezolid-resistant strains.

Detection of Staphylococcus aureus isolates with heterogeneous intermediate-level resistance to vancomycin in the United States.

The prevalence of heterogeneous intermediate-level resistance to vancomycin (hVISA) in Staphylococcus aureus was assessed by screening a large collection of recent isolates. Susceptibility testing by the Clinical and Laboratory Standards Institute broth microdilution method and the Etest GRD (glycopeptide resistance detection) method (bioMérieux) was performed on 4,210 clinically significant S. aureus isolates obtained in 2009 from 43 U.S. centers. Isolates with Etest GRD-positive results for hVISA were evaluated further by repeat GRD testing and population analysis profiling-area under the curve (PAP-AUC) analysis. No VISA (vancomycin MIC, 4 to 8 µg/ml) or vancomycin-resistant (MIC ≥ 16 µg/ml) strains were detected. The Etest GRD screen for hVISA was initially positive for 68 isolates (1.6%; all by teicoplanin MIC ≥ 8 µg/ml at 24 or 48 h). Among those 68 isolates, 45 were reproducibly GRD positive. PAP-AUC testing confirmed only 11 isolates as hVISA (all had reproducible GRD-positive results). The 11 hVISA isolates were from nine medical centers and appeared genetically diverse (ten different PFGE types). The rates of resistance (including intermediate) for hVISA were as follows: oxacillin, 82%; erythromycin, 82%; clindamycin, 73%; levofloxacin, 73%; trimethoprim-sulfamethoxazole, 9%; and daptomycin, 9%. All hVISA isolates were susceptible to linezolid, tigecycline, and ceftaroline. Our data suggest that the overall prevalence of hVISA in the United States is low (0.3%). The hVISA isolates represented 10.5% of isolates with vancomycin MICs of 2 µg/ml and 0.1% of isolates with vancomycin MICs of 1 µg/ml. The positive predictive value of GRD Etest for hVISA was 16.2% for initial screen positive and 24.4% for reproducibly positive results.

Changing epidemiology of antimicrobial-resistant Streptococcus pneumoniae in the United States, 2004-2005.

BACKGROUND: The impact of pediatric 7-valent pneumococcal conjugate vaccination (PCV-7) on the population of Streptococcus pneumoniae in the United States was examined by determining the serotypes, antimicrobial resistance profiles, and genetic relatedness of isolates from patients with invasive and noninvasive infections during the 2004-2005 respiratory illness season. METHODS: Susceptibility testing, serotyping, and pulsed-field gel electrophoresis analysis were performed on 1647 S. pneumoniae isolates obtained from 41 US medical centers in 2004-2005 as part of a longitudinal antimicrobial resistance surveillance program. The results were compared with surveillance data from earlier periods. RESULTS: From the 1999-2000 to the 2004-2005 respiratory illness season, the prevalence of isolates with intermediate penicillin resistance (minimum inhibitory concentration, 0.1-1 microg/mL) increased from 12.7% to 17.9%, prevalence of penicillin-resistant isolates (minimum inhibitory concentration, >or=2 microg/mL) decreased from 21.5% to 14.6%, and prevalence of isolates resistant to erythromycin increased from 25.7% to 29.1% among S. pneumoniae isolates. The prevalence of multidrug resistance among isolates did not change (22.4% in 1999-2000 and 20.0% in 2004-2005). Sixty different serotypes were recognized among the isolates from 2004-2005; predominant serotypes were 19A (14.5%), 3 (11.2%), 6A (7.1%), 19F (7%), and 11A (6%). Serotypes that were included in PCV-7 accounted for 16.3% of isolates; 28.4% of strains isolated had PCV-7-related serotypes, and the remaining 55.3% of isolates had serotypes that were unrelated to PCV-7. The serotype distribution of the penicillin-resistant S. pneumoniae population changed from 1999-2000 to 2004-2005, with an increase in the prevalence of serotype 19A (1.5% to 35.4%) and serotype 35B (1.2% to 12.5%) and a decrease in the prevalence of most PCV-7 serotypes, including 23F (16.1% to 5%), 9V (16.1% to 4.2%), 6B (13.7% to 3.8%), and 14 (18.5% to 2.9%). CONCLUSIONS: The penicillin-resistant S. pneumoniae population has changed; most isolates are now closely related to 2 Pneumococcal Molecular Epidemiology Network clones that increased in prevalence from 1999-2000 to 2004-2005 (Taiwan(19F)-14 [14.6% to 36.7%; 60% were serotype 19A] and Utah(35B)-24 [0.9% to 16.3%]).

Community-associated methicillin-resistant Staphylococcus aureus, Iowa, USA.

We performed antimicrobial drug susceptibility testing and molecular typing on invasive methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 1,666) submitted to the University of Iowa Hygienic Laboratory during 1999-2006 as part of a statewide surveillance system. All USA300 and USA400 isolates were resistant to

BD Phoenix and Vitek 2 detection of mecA-mediated resistance in Staphylococcus aureus with cefoxitin.

The BD Phoenix (BD Diagnostics, Sparks, MD) and Vitek 2 (bioMérieux, Durham, NC) automated susceptibility testing systems have implemented the use of cefoxitin to enhance the detection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). To assess the impact of this change, 620 clinically significant S. aureus isolates were tested in parallel on Phoenix PMIC/ID-102 panels and Vitek 2 AST-GP66 cards. The results for oxacillin and cefoxitin generated by the automated systems were compared to those generated by two reference methods: mecA gene detection and MICs of oxacillin previously determined by broth microdilution according to CLSI guidelines. Testing of isolates with discordant results was repeated to attain a majority or consensus final result. There was 100% final agreement between the results of the two reference methods. For the 448 MRSA and 172 methicillin-susceptible S. aureus isolates tested, the rates of categorical agreement of the results obtained with the automated systems with those obtained by the reference methods were 99.8% for the Phoenix panels and 99.7% for the Vitek 2 cards. A single very major error occurred on each instrument (0.2%) with different MRSA isolates. The only major error was attributed to the Vitek 2 system overcalling oxacillin resistance. In 16 instances (9 on the Phoenix system, 7 on the Vitek 2 system), an oxacillin MIC in the susceptible range was correctly changed to resistant by the expert system on the basis of the cefoxitin result. The inclusion of cefoxitin in the Phoenix and Vitek 2 panels has optimized the detection of MRSA by both systems.

Quorum Sensing, Virulence, and Antibiotic Resistance of USA100 Methicillin-Resistant Staphylococcus aureus Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) infections impact all patient populations both in the community and in health care settings. Despite advances in our knowledge of MRSA virulence, little is known about the regulatory mechanisms of USA100 health care-associated MRSA isolates, which are the second most frequently identified MRSA isolates found in all infections. This work focused on the contribution of the USA100 agr type II quorum-sensing system to virulence and antibiotic resistance. From a MRSA strain collection, we selected 16 representative USA100 isolates, constructed mutants with Δagr mutations, and characterized selected strain pairs for virulence factor expression, murine skin infection, and antibiotic resistance. For each strain pair, hemolysis and extracellular protease expression were significantly greater in the wild-type (WT) strains than in the Δagr mutants. Similarly, mice challenged with the WT strains had larger areas of dermonecrosis and greater weight loss than those challenged with the Δagr mutants, demonstrating that the USA100 agr system regulates virulence. Although USA100 isolates exhibit a high level of antibiotic resistance, the WT and Δagr strain pairs showed no difference in MICs by MIC testing. However, in the presence of a sub-MIC of vancomycin, most of the USA100 Δagr mutants exhibited slower growth than the WT isolates, and a couple of the Δagr mutants also grew more slowly in the presence of a sub-MIC of cefoxitin. Altogether, our findings demonstrate that the USA100 agr system is a critical regulator of virulence, and it may have a contribution to the optimal survival of these MRSA strains in the presence of antibiotics.IMPORTANCE USA100 health care-associated MRSA isolates are highly antibiotic resistant and can cause invasive disease across all patient populations. Even though USA100 strains are some of the most frequently identified causes of infections, little is known about virulence regulation in these isolates. Our study demonstrates that the USA100 agr quorum-sensing system is important for the control of toxin and exoenzyme production and that the agr system has a key role in skin infection. In some USA100 isolates, the agr system is important for growth in the presence of low levels of antibiotics. Altogether, our findings demonstrate that the USA100 agr system is a critical regulator of virulence and that it may make a contribution to the optimal survival of these MRSA strains in the presence of antibiotics.

Accuracy of phenotypic methods for identification of Streptococcus pneumoniae isolates included in surveillance programs.

Similarities between Streptococcus pneumoniae and viridans group streptococci may result in misidentification of these organisms. In surveillance programs which assess antimicrobial resistance rates among respiratory tract pathogens, such identification errors could lead to overestimates of pneumococcal resistance rates. DNA probe analysis (Gen-Probe, San Diego, CA), the bile solubility test, optochin susceptibility, colony morphology, and the capsular swelling reaction with Omni serum (Staten Serum Institut, Copenhagen, Denmark) were used to characterize 1,733 organisms provisionally identified as S. pneumoniae in a 2004 to 2005 antimicrobial resistance surveillance program. These organisms were obtained in 41 U.S. medical centers. Among these, 1,647 (95%) were determined to be S. pneumoniae by DNA probe. Elimination of those isolates found not to be S. pneumoniae resulted in 1 to 2% decreases in resistance rate estimates with penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. With AccuProbe as a reference standard, the sensitivities and specificities of each phenotypic method for the identification of S. pneumoniae were, respectively, 98.8% and 82.6% for bile solubility, 99.3% and 74.4% for the capsular swelling reaction with Omni serum, and 87.9% and 59.3% for optochin susceptibility. Colony morphology was of limited value, as 391 (23.7%) isolates lacked the typical button or mucoid colony appearance of S. pneumoniae.

Increasing telithromycin resistance among Streptococcus pyogenes in Europe.

OBJECTIVES: To assess changes in macrolide and ketolide resistance among Streptococcus pyogenes in Europe and to examine the relationship of resistance to antimicrobial usage. METHODS: Clinical S. pyogenes isolates were collected from Denmark, Finland, France, Germany, Italy, Netherlands, Norway, Spain, Sweden, UK, Croatia, Hungary, Poland, Slovak Republic and Slovenia during 2002-03 (n = 2165) and 2004-05 (n = 2333). Resistance to telithromycin (MIC > or = 2) and erythromycin (MIC > or = 0.5) was determined by CLSI broth microdilution. Changes in resistance over time and the relationship of resistance to antimicrobial use (European Surveillance of Antimicrobial Consumption data) were assessed. Telithromycin-resistant isolates were characterized by PFGE to determine genetic relatedness and by PCR to detect mef(A), erm(A) and erm(B). RESULTS: The erythromycin resistance rate during 2004-05 (11.6%) was similar to 2002-03 (10.4%). The proportion of macrolide-resistant isolates with the constitutive MLS(B) phenotype increased from 29.3% (2002-03) to 45.7% (2004-05). Telithromycin resistance increased from 1.8% in 2002-03 to 5.2% in 2004-05. For Western Europe, associations of telithromycin and erythromycin resistance, respectively, were found with azithromycin use (R2 = 0.52 and 0.60), clarithromycin use (R2 = 0.76 and 0.85) and total macrolide/lincosamide use (R2 = 0.75 and 0.69). For Eastern Europe, associations of antimicrobial use with resistance were not apparent. The 162 telithromycin-resistant isolates comprised 42 PFGE patterns with 68.5% in eight major PFGE groups. The erm(B) gene was detected in 155 of the 162 telithromycin-resistant isolates. CONCLUSIONS: Significant increases in telithromycin resistance occurred from 2002-03 to 2004-05 in Europe. Macrolide use appears to be a factor in the emergence of ketolide resistance among S. pyogenes in Western Europe.

Redefining the Chronic-Wound Microbiome: Fungal Communities Are Prevalent, Dynamic, and Associated with Delayed Healing.

UNLABELLED: Chronic nonhealing wounds have been heralded as a silent epidemic, causing significant morbidity and mortality especially in elderly, diabetic, and obese populations. Polymicrobial biofilms in the wound bed are hypothesized to disrupt the highly coordinated and sequential events of cutaneous healing. Both culture-dependent and -independent studies of the chronic-wound microbiome have almost exclusively focused on bacteria, omitting what we hypothesize are important fungal contributions to impaired healing and the development of complications. Here we show for the first time that fungal communities (the mycobiome) in chronic wounds are predictive of healing time, associated with poor outcomes, and form mixed fungal-bacterial biofilms. We longitudinally profiled 100, nonhealing diabetic-foot ulcers with high-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus, estimating that up to 80% of wounds contain fungi, whereas cultures performed in parallel captured only 5% of colonized wounds. The "mycobiome" was highly heterogeneous over time and between subjects. Fungal diversity increased with antibiotic administration and onset of a clinical complication. The proportions of the phylum Ascomycota were significantly greater (P = 0.015) at the beginning of the study in wounds that took >8 weeks to heal. Wound necrosis was distinctly associated with pathogenic fungal species, while taxa identified as allergenic filamentous fungi were associated with low levels of systemic inflammation. Directed culturing of wounds stably colonized by pathogens revealed that interkingdom biofilms formed between yeasts and coisolated bacteria. Combined, our analyses provide enhanced resolution of the mycobiome during impaired wound healing, its role in chronic disease, and impact on clinical outcomes. IMPORTANCE: Wounds are an underappreciated but serious complication for a diverse spectrum of diseases. High-risk groups, such as persons with diabetes, have a 25% lifetime risk of developing a wound that can become chronic. The majority of microbiome research related to chronic wounds is focused on bacteria, but the association of fungi with clinical outcomes remains to be elucidated. Here we describe the dynamic fungal communities in 100 diabetic patients with foot ulcers. We found that communities are unstable over time, but at the first clinical presentation, the relative proportions of different phyla predict healing times. Pathogenic fungi not identified by culture reside in necrotic wounds and are associated with a poor prognosis. In wounds stably colonized by fungi, we identified yeasts capable of forming biofilms in concert with bacteria. Our findings illuminate the associations of the fungal mycobiome with wound prognosis and healing.

Airway acidification initiates host defense abnormalities in cystic fibrosis mice.

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. In humans and pigs, the loss of CFTR impairs respiratory host defenses, causing airway infection. But CF mice are spared. We found that in all three species, CFTR secreted bicarbonate into airway surface liquid. In humans and pigs lacking CFTR, unchecked H(+) secretion by the nongastric H(+)/K(+) adenosine triphosphatase (ATP12A) acidified airway surface liquid, which impaired airway host defenses. In contrast, mouse airways expressed little ATP12A and secreted minimal H(+); consequently, airway surface liquid in CF and non-CF mice had similar pH. Inhibiting ATP12A reversed host defense abnormalities in human and pig airways. Conversely, expressing ATP12A in CF mouse airways acidified airway surface liquid, impaired defenses, and increased airway bacteria. These findings help explain why CF mice are protected from infection and nominate ATP12A as a potential therapeutic target for CF.

Antimicrobial resistance among Streptococcus pneumoniae in the United States: have we begun to turn the corner on resistance to certain antimicrobial classes?

BACKGROUND: Antimicrobial resistance has emerged as a major problem in Streptococcus pneumoniae in the United States during the past 15 years. This study was undertaken to elucidate the current scope and magnitude of this problem in the United States and to assess resistance trends since 1994-1995. METHODS: A total of 1817 S. pneumoniae isolates obtained from patients with community-acquired respiratory tract infections in 44 US medical centers were characterized during the winter of 2002-2003. The activity of 27 antimicrobial agents was assessed. In addition, selected isolates were examined for the presence of mutations in the quinolone-resistance determining regions (QRDRs) of parC and gyrA that resulted in diminished fluoroquinolone activity. The results of this survey were compared with the results of 4 previous surveys conducted in a similar manner since 1994-1995. RESULTS: Overall rates of resistance (defined as the rate of intermediate resistance plus the rate of resistance) were as follows: penicillin, 34.2%; ceftriaxone, 6.9%; erythromycin, 29.5%; clindamycin, 9.4%; tetracycline, 16.2%; and trimethoprim-sulfamethoxazole (TMP-SMX), 31.9%. No resistance was observed with vancomycin, linezolid, or telithromycin; 22.2% of isolates were multidrug resistant; 2.3% of isolates had ciprofloxacin MICs of >or=4.0 microg/mL. It was estimated that 21.9% of the isolates in this national collection had mutations in the QRDRs of parC and/or gyrA, with parC only mutations occurring most often (in 21% of all isolates). Trend analysis since 1994-1995 indicated that rates of resistance to beta -lactams, macrolides, tetracyclines, TMP-SMX, and multiple drugs have either plateaued or have begun to decrease. Conversely, fluoroquinolone resistance among S. pneumoniae is becoming more prevalent. CONCLUSION: It appears that, as fluoroquinolone resistance emerges among S. pneumoniae in the United States, resistance to other antimicrobial classes is becoming less common.

Macrolide-resistant Streptococcus pyogenes in the United States, 2002-2003.

BACKGROUND: Increased levels of macrolide-resistant Streptococcus pyogenes in focal regions of the United States have been reported. The purpose of this study was to determine the antimicrobial susceptibility of a large collection of S. pyogenes isolates from throughout the United States and to elucidate the mechanisms of resistance and genetic relatedness of macrolide-resistant isolates. METHODS: During 2002-2003, a total of 1885 S. pyogenes clinical isolates were obtained from 45 US medical centers. Susceptibility to penicillin, cefdinir, erythromycin, azithromycin, clarithromycin, clindamycin, telithromycin, and levofloxacin was determined. Macrolide resistance phenotypes were determined by double-disk diffusion, and macrolide resistance genotypes were determined by polymerase chain reaction and sequencing. All macrolide-resistant isolates and all isolates recovered from sterile sites were further characterized by pulsed-field gel electrophoresis (PFGE) and emm typing. RESULTS: The majority (85%) of isolates were pharyngeal. Resistance was detected to erythromycin (6.8% of isolates), azithromycin (6.9%), clarithromycin (6.6%), clindamycin (0.5%), telithromycin (0.2%), and levofloxacin (0.05%). The macrolide-resistance phenotype distribution was as follows: macrolide-lincosamide-streptogramin B (MLSB), 56% of isolates (inducible, 47%; constitutive, 9%); and M, 44%. The genotypes detected were as follows: ermA, 46% of isolates (95% with inducible MLSB phenotype); mefA, 43% (all with M phenotype); and ermB, 8.5% (45% with inducible MLSB and 45% with constitutive MLSB). Three isolates with constitutive MLSB phenotypes had 23S ribosomal RNA mutations. The 129 erythromycin-resistant isolates belonged to 28 emm types and 44 PFGE patterns, with 51% of the isolates in 4 major PFGE clones each associated with a predominant emm type (emm75, emm58, emm12, and emm114) and resistance genotype (mefA or ermA)). CONCLUSIONS: The population of macrolide-resistant S. pyogenes isolates in the United States is small, but it includes several large clones with potential for expansion.

Castanea sativa (European Chestnut) Leaf Extracts Rich in Ursene and Oleanene Derivatives Block Staphylococcus aureus Virulence and Pathogenesis without Detectable Resistance.

The Mediterranean is home to a rich history of medical traditions that have developed under the influence of diverse cultures over millennia. Today, many such traditions are still alive in the folk medical practices of local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections led us to study Castanea sativa (European Chestnut) for its potential antibacterial activity. Here, we report the quorum sensing inhibitory activity of refined and chemically characterized European Chestnut leaf extracts, rich in oleanene and ursene derivatives (pentacyclic triterpenes), against all Staphylococcus aureus accessory gene regulator (agr) alleles. We present layers of evidence of agr blocking activity (IC50 1.56-25 µg mL-1), as measured in toxin outputs, reporter assays hemolytic activity, cytotoxicity studies, and an in vivo abscess model. We demonstrate the extract's lack of cytotoxicity to human keratinocytes and murine skin, as well as lack of growth inhibitory activity against S. aureus and a panel of skin commensals. Lastly, we demonstrate that serial passaging of the extract does not result in acquisition of resistance to the quorum quenching composition. In conclusion, through disruption of quorum sensing in the absence of growth inhibition, this study provides insight into the role that non-biocide inhibitors of virulence may play in future antibiotic therapies.

Cultures of diabetic foot ulcers without clinical signs of infection do not predict outcomes.

OBJECTIVE: We examined associations between ulcer bioburden and ulcer outcomes in neuropathic diabetic foot ulcers (DFUs) that lacked clinical signs of infection. RESEARCH DESIGN AND METHODS: Three dimensions of bioburden (i.e., microbial load, microbial diversity, and the presence of likely pathogens) were measured at baseline using swab cultures obtained by Levine's technique. Subjects were assessed every 2 weeks for 26 weeks to determine the rate of healing and development of infection-related complications. Foot ulcers were off-loaded using total-contact casts and routinely debrided. To establish associations between bioburden and rate of healing, Cox proportional hazards and least squares regression were used after adjusting for ulcer depth, surface area, and duration. RESULTS: A total of 77 subjects completed the study. Sixty-five (84.4%) had ulcers that healed during follow-up; weeks-to-closure ranged from 2 to 26 (median 4.0). Mean (± SD) percent reduction in surface area/week was 25.0% (± 23.33). Five (6.5%) of the DFUs developed an infection-related complication. None of the bioburden dimensions (i.e., microbial load, microbial diversity, or presence of likely pathogens) was significantly associated with weeks-to-closure or percent reduction in surface area per week. Weeks-to-closure was best predicted by ulcer duration, depth, and surface area (c-statistic = 0.75). CONCLUSIONS: Culturing DFUs that showed no clinical signs of infection had no predictive value for outcomes of DFUs managed with total-contact casts and routine debridement. These findings support recommendations of the Infectious Disease Society of America that culturing and antibiotics should be avoided in treating DFUs that show no clinical signs of infection.