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.
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.
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.
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-β-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.
The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is changing, with USA300 emerging first in community and then in healthcare settings. We performed nationwide surveillance to ...assess recent trends in the molecular epidemiology of MRSA.
One hundred consecutive unique clinically significant S. aureus isolates were recovered from patients at each of 43 US centers between July 1, 2011, and December 31, 2011. Susceptibility testing, pulsed-field gel electrophoresis (PFGE), staphylococcal protein A gene (spa) and staphylococcal cassette chromosome mec typing, and Panton-Valentine leukocidin detection were performed on all MRSA isolates.
Of 4,131 isolates collected, 2,093 (51%) were MRSA. Specimen sources of MRSA isolates included wound or abscess (54%), blood (24%), lower respiratory tract (11%), and other sterile site (10%). Thirty percent were isolated more than 48 hours after hospital admission (ie, were associated with nosocomial acquisition of infection). USA300 was the most common PFGE type (1,269 isolates; 61%), overall and in all regions, followed by USA100 (368 isolates; 18%). Among 173 spa types found, the most common were t008 (51%) and t002 (18%); no other spa type accounted for more than 2% of isolates. One strain type (USA300/t008/IV) constituted almost half of all MRSA isolates (1,005 isolates; 48%) and was the most common at all body sites, causing 37% of MRSA bloodstream infections (BSIs) and 38% of nosocomial MRSA infections. Multidrug-resistant phenotypes were found among 34 USA300 isolates (3%) from 18 states.
The USA300 PFGE type continues to advance nationwide. A single strain type (USA300/t008/IV) predominates in all regions and infection sites and is now more common than USA100 as a cause of MRSA BSI and nosocomial infections. Although most USA300 retain typical susceptibility profiles, multidrug-resistant phenotypes are emerging.
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 µg/mL) increased from 12.7% to 17.9%, prevalence of penicillin-resistant isolates (minimum inhibitory concentration, ⩾2 µg/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 (Taiwan19F-14 14.6% to 36.7%; 60% were serotype 19A and Utah35B-24 0.9% to 16.3%).
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
(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 IC
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
Finally, we employed mass spectrometry to identify or confirm the structure of quorum sensing signaling peptides in three strains each of
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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.
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