Leveraging the human microbiota to target bacterial respiratory pathogens: new paths toward an expanded antimicrobial armamentarium.

Acute respiratory infections are the most frequent infections across the lifespan and are the leading infectious cause of death among children globally. Bacterial respiratory infections are routinely treated with antibiotics, nearly all of which are derived from microbial natural products. Unfortunately, antibiotic-resistant bacteria are an increasingly frequent cause of respiratory infections, and there are few new antibiotics in development that target these pathogens. In the article by Stubbendieck et al., the authors identified Rothia species that demonstrate in vitro and ex vivo growth inhibition of the respiratory pathobiont Moraxella catarrhalis. The authors present experiments suggesting that this activity is mediated at least in part through the secretion of a novel peptidoglycan endopeptidase that targets the M. catarrhalis cell wall. In this commentary, we discuss these findings in the context of the urgent threat of antimicrobial resistance and highlight the promise of the human respiratory microbiota as a source of novel biotherapeutics.

[Current microbiological aspects of community respiratory infection beyond COVID-19]. / Aspectos microbiológicos actuales de la infección respiratoria comunitaria más allá de la COVID-19.

From a microbiological point of view, both empirical and targeted antimicrobial treatment in respiratory infection is based on the sensitivity profile of isolated microorganisms and the possible resistance mechanisms that they may present. The latter may vary in different geographic areas according to prescription profiles and vaccination programs. Beta-lactam antibiotics, fluoroquinolones, and macrolides are the most commonly used antimicrobials during the exacerbations of chronic obstructive pulmonary disease and community-acquired pneumonia. In their prescription, different aspects such as intrinsic activity, bactericidal effect or their ability to prevent the development of resistance must be taken into account. The latter is related to the PK/PD parameters, the mutant prevention concentration and the so-called selection window. More recently, the potential ecological impact has grown in importance, not only on the intestinal microbiota, but also on the respiratory one. Maintaining the state of eubiosis requires the use of antimicrobials with a low profile of action on anaerobic bacteria. With their use, the resilience of the bacterial populations belonging to the microbiota, the state of resistance of colonization and the collateral damage related to the emergence of resistance to the antimicrobials in pathogens causing the infections and in the bacterial populations integrating the microbiota.

Aluminum-Substituted Keggin Germanotungstate [HAl(H2O)GeW11O39]4-: Synthesis, Characterization, and Antibacterial Activity.

We report on the new monosubstituted aluminum Keggin-type germanotungstate (C4H12N)4[HAlGeW11O39(H2O)]·11H2O ([Al(H2O)GeW11]4-), which has been synthesized at room temperature via rearrangement of the dilacunary [γ-GeW10O36]8- polyoxometalate precursor. [Al(H2O)GeW11]4- has been characterized thoroughly both in the solid state by single-crystal and powder X-ray diffraction, IR spectroscopy, thermogravimetric analysis, and elemental analysis as well as in solution by cyclic voltammetry (CV) 183W, 27Al NMR and UV-vis spectroscopy. A study on the antibacterial properties of [Al(H2O)GeW11]4- and the known aluminum(III)-centered Keggin polyoxotungstates (Al-POTs) α-Na5[AlW12O40] (α-[AlW12O40]5-) and Na6[Al(AlOH2)W11O39] ([Al(AlOH2)W11O39]6-) revealed enhanced activity for all three Al-POTs against the Gram-negative bacterium Moraxella catarrhalis (minimum inhibitory concentration (MIC) up to 4 µg mL-1) and the Gram-positive Enterococcus faecalis (MIC up to 128 µg mL-1) compared to the inactive Al(NO3)3 salt (MIC > 256 µg mL-1). CV indicates the redox activity of the Al-POTs as a dominating factor for the observed antibacterial activity with increased tendency to reduction, resulting in increased antibacterial activity of the POT.

Antibacterial Fusion Proteins Enhance Moraxella catarrhalis Killing.

Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis. These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis, therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens.

Molecular Networking-Guided Discovery and Characterization of Stechlisins, a Group of Cyclic Lipopeptides from a Pseudomonas sp.

Increasingly sensitive analytical instruments and robust downstream data processing tools have revolutionized natural product research over the past decade. A molecular networking-guided survey led to the identification of 33 new cyclic lipopeptides (CLPs) from the culture broth of the proteobacterium Pseudomonas sp. FhG100052. The compound family resembles members of the amphisin group of CLPs that possess a 3-hydroxy fatty acid linked to the N-terminus of an undecapeptide core. Culture optimization led to the isolation and subsequent structure elucidation of one known and five new derivatives by extensive MS/MS and NMR experiments in combination with Marfey's analysis. The data were in agreement with in silico analysis of the corresponding biosynthetic gene cluster. Most strikingly, the length of the incorporated fatty acid defined the growth inhibitory effects against Moraxella catarrhalis FH6810, as observed by MIC values ranging from no inhibition (>128 µg/mL) to 4 µg/mL.

Antimicrobial Photodynamic Therapy with Chlorin e6 Is Bactericidal against Biofilms of the Primary Human Otopathogens.

Moraxella catarrhalis, Streptococcus pneumoniae, and nontypeable Haemophilus influenzae (NTHi) are ubiquitous upper respiratory opportunistic pathogens. Together, these three microbes are the most common causative bacterial agents of pediatric otitis media (OM) and have therefore been characterized as the primary human otopathogens. OM is the most prevalent bacterial infection in children and the primary reason for antibiotic administration in this population. Moreover, biofilm formation has been confirmed as a primary mechanism of chronic and recurrent OM disease. As bacterial biofilms are inherently metabolically recalcitrant to most antibiotics and these complex structures also present a significant challenge to the immune system, there is a clear need to identify novel antimicrobial approaches to treat OM infections. In this study, we evaluated the potential efficacy of antibacterial photodynamic therapy (aPDT) with the photosensitizer chlorin e6 (Ce6) against planktonic as well as biofilm-associated M. catarrhalis, S. pneumoniae, and NTHi. Our data indicate aPDT with Ce6 elicits significant bactericidal activity against both planktonic cultures and established biofilms formed by the three major otopathogens (with an efficacy of ≥99.9% loss of viability). Notably, the implementation of a novel, dual-treatment aPDT protocol resulted in this disinfectant effect on biofilm-associated bacteria and, importantly, inhibited bacterial regrowth 24 h posttreatment. Taken together, these data suggest this novel Ce6-aPDT treatment may be a powerful and innovative therapeutic strategy to effectively treat and eradicate bacterial OM infections and, significantly, prevent the development of recurrent disease.IMPORTANCE Otitis media (OM), or middle ear disease, is the most prevalent bacterial infection in children and the primary reason for antibiotic use and surgical intervention in the pediatric population. Biofilm formation by the major bacterial otopathogens, Moraxella catarrhalis, Streptococcus pneumoniae, and nontypeable Haemophilus influenzae, has been shown to occur within the middle ears of OM patients and is a key factor in the development of recurrent disease, which may result in hearing impairment and developmental delays. Bacterial biofilms are inherently impervious to most antibiotics and present a significant challenge to the immune system. In this study, we demonstrate that antimicrobial photodynamic therapy (aPDT) using the photosensitizer chlorin e6 elicits significant bactericidal activity versus planktonic and biofilm-associated otopathogens and supports further analyses of this novel, efficacious, and promising technology as an adjunctive treatment for acute and recurrent OM.

Bactericidal activity of hexylresorcinol lozenges against oropharyngeal organisms associated with acute sore throat.

OBJECTIVE: For the majority of people with acute sore throat, over-the-counter treatments represent the primary option for symptomatic relief. This study evaluated the in vitro bactericidal activity of lozenges containing the antiseptic hexylresorcinol against five bacteria associated with acute sore throat: Staphylococcus aureus, Streptococcus pyogenes, Moraxella catarrhalis, Haemophilus influenzae and Fusobacterium necrophorum. RESULTS: Hexylresorcinol 2.4 mg lozenges were dissolved into 5 mL of artificial saliva medium. Inoculum cultures were prepared in triplicate for each test organism to give an approximate population of 108 colony-forming units (cfu)/mL. Bactericidal activity was measured by log reduction in cfu. Greater than 3log10 reductions in cfu were observed at 1 min after dissolved hexylresorcinol lozenges were added to S. aureus (log10 reduction cfu/mL ± standard deviation, 3.3 ± 0.2), M. catarrhalis (4.7 ± 0.4), H. influenzae (5.8 ± 0.4) and F. necrophorum (4.5 ± 0.2) and by 5 min for S. pyogenes (4.3 ± 0.4). Hexylresorcinol lozenges achieved a > 99.9% reduction in cfu against all tested organisms within 5 min, which is consistent with the duration for a lozenge to dissolve in the mouth. In conclusion, in vitro data indicate that hexylresorcinol lozenges offer rapid bactericidal activity against organisms implicated in acute sore throat.

Photoinactivation of Moraxella catarrhalis Using 405-nm Blue Light: Implications for the Treatment of Otitis Media.

Moraxella catarrhalis is one of the major otopathogens of otitis media (OM) in childhood. M. catarrhalis tends to form biofilm, which contributes to the chronicity and recurrence of infections, as well as resistance to antibiotic treatment. In this study, we aimed to investigate the effectiveness of antimicrobial blue light (aBL; 405 nm), an innovative nonpharmacological approach, for the inactivation of M. catarrhalis OM. M. catarrhalis either in planktonic suspensions or 24-h old biofilms were exposed to aBL at the irradiance of 60 mW cm-2 . Under an aBL exposure of 216 J cm-2 , a >4-log10 colony-forming units (CFU) reduction in planktonic suspensions and a >3-log10 CFU reduction in biofilms were observed. Both transmission electron microscopy and scanning electron microscopy revealed aBL-induced morphological damage in M. catarrhalis. Ultraperformance liquid chromatography results indicated that protoporphyrin IX and coproporphyrin were the two most abundant species of endogenous photosensitizing porphyrins. No statistically significant reduction in the viability of HaCaT cells was observed after an aBL exposure of up to 216 J cm-2 . Collectively, our results suggest that aBL is potentially an effective and safe alternative therapy for OM caused by M. catarrhalis. Further in vivo studies are warranted before this optical approach can be moved to the clinics.

Co-carriage of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis among three different age categories of children in Hungary.

BACKGROUND: The nasopharynx can from time to time accommodate otherwise pathogenic bacteria. This phenomenon is called asymptomatic carriage. However, in case of decreased immunity, viral infection or any other enhancing factors, severe disease can develop. Our aim in this study was to survey the nasal carriage rates of four important respiratory pathogens in three different age groups of children attending nurseries, day-care centres and primary schools. This is the first study from Hungary about the asymptomatic carriage of H. influenzae and M. catarrhalis. METHODS: Altogether 580 asymptomatic children were screened in three Hungarian cities. Samples were collected from both nostrils with cotton swabs. The identification was based on both colony morphology and species-specific PCRs. Serotyping was performed for S. pneumoniae, H. influenzae and M. catarrhalis. Antibiotic susceptibility was determined with agar dilution, according to the EUCAST guidelines. Clonality was examined by PFGE. RESULTS AND CONCLUSIONS: Whereas the carriage rates of S. pneumoniae, H. influenzae and M. catarrhalis clearly decreased with age, that of S. aureus showed an opposite tendency. Multiple carriage was least prevalent if S. aureus was one of the participants. The negative association between this bacterium and the others was statistically significant. For pneumococcus, the overall carriage rate was lower compared to earlier years, and PCV13 serotypes were present in only 6.2% of the children. The majority of H. influenzae isolates was non-typeable and no type b was detected; serotype A was dominant among M. catarrhalis. All four bacteria were more sensitive to antibiotics compared to clinical isolates. No MRSAs were detected, but we found three mupirocin resistant strains. The positive effect of Hib- and PCV-vaccination is undoubted. Continued surveillance of these pathogens is required.

Respiratory pathogens - Some altered antibiotic susceptibility after implementation of pneumococcus vaccine and antibiotic control strategies.

BACKGROUND/PURPOSE: Antimicrobial resistance in Taiwan has been on the rise for two decades. The implementation of pneumococcal conjugate vaccination (PCV13) and enhanced antimicrobial control (2013-2015) by the government may have changed the antibiotic resistance. METHODS: Four respiratory pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, and Moraxella catarrhalis) isolated in a single medical center during 2008-2017 were studied. We defined three temporal stages: (a) the first era (2008-2012), prior to implementation of the national immunization program (PCV13 vaccination), (b) the second era (2013-2015), during which an enhanced antibiotic control strategy was implemented, and (c) the third era (2016-2017), after implementation. Antimicrobial drug sensitivities were collected from two other hospitals: one from east Taiwan, one from west-central Taiwan. RESULTS: S. pneumoniae was frequently isolated during the first era. It declined progressively during the second era of PCV13 vaccination. S. pyogenes and M. catarrhalis were not frequently isolated. The drug susceptibility of S. pneumoniae to ceftriaxone and vancomycin remained high. The antimicrobial susceptibility of H. influenzae to amoxillin/clavulante declined over the three temporal stages, from 91.9%-79.5%-58.5% (all p < 0.05). CONCLUSION: Antimicrobial resistance of H. influenzae increased during the latter part of the study period. The PCV13 vaccination program reduced the invasive pneumococcal disease and reduced the stress on the emergent drug resistance. This enhanced antibiotic control strategy was effective in terms of nosocomial drug resistance but not for community-associated pathogens.

C4-Phenylthio ß-lactams: Effect of the chirality of the ß-lactam ring on antimicrobial activity.

C4-phenylthio ß-lactams are a new family of antibacterial agents that have activity against two phylogenetically distant bacteria - Mycobacterium tuberculosis (Mtb) and Moraxella catarrhalis (M. cat). These compounds are effective against ß-lactamase producing Mtb and M. cat unlike the clinically relevant ß-lactam antibiotics. The structure-activity relationship for the C4 phenylthio ß-lactams has not yet been completely defined. Earlier efforts in our laboratories established that the C4-phenylthio substituent is essential for antimicrobial activity, while the N1 carbamyl substituent plays a more subtle role. In this present study, we investigated the role that the stereochemistry at C4 plays in these compounds' antibacterial activity. This was achieved by synthesizing and testing the antimicrobial activity of diastereomers with a chiral carbamyl group at N1. Our findings indicate that a strict stereochemistry for the C4-phenylthio ß-lactams is not required to obtain optimal anti-Mtb and anti-M. cat activity. Furthermore, the structure-bioactivity profiles more closely relate to the electronic requirement of the phenylthiogroup. In addition, the MICs of Mtb are sensitive to growth medium composition. Select compounds showed activity against non-replicating and multi-drug resistant Mtb.

Antimicrobial susceptibility and impact of macrolide antibiotics on Moraxella catarrhalis in the upper and lower airways of children with chronic endobronchial suppuration.

INTRODUCTION: Moraxella catarrhalis is an important but insufficiently studied respiratory pathogen. AIM: To determine antibiotic susceptibility and impact of recent antibiotics on M. catarrhalis from children with chronic endobronchial suppuration. METHODOLOGY: We cultured nasopharyngeal (NP) swabs and bronchoalveolar lavage (BAL) fluids collected from children who were prospectively enrolled in studies of chronic cough and had flexible bronchoscopy performed. Recent ß-lactam or macrolide antibiotic use was recorded. M. catarrhalis isolates stored at -80 °C were re-cultured and susceptibility determined to a range of antibiotics including the macrolide antibiotic erythromycin. RESULTS: Data from concurrently collected NP and BAL specimens were available from 547 children (median age 2.4 years) enrolled from 2007 to 2016. M. catarrhalis NP carriage was detected in 149 (27  %) children and lower airway infection (≥104 c.f.u. ml-1 BAL) in 67 (12  %) children. In total, 91  % of 222 M. catarrhalis isolates were ß-lactamase producers, and non-susceptibility was high to benzylpenicillin (98 %), cefaclor (39 %) and cotrimoxazole (38 %). Overall, >97  % isolates were susceptible to cefuroxime, chloramphenicol, erythromycin and tetracycline; three isolates were erythromycin-resistant (MIC >0.5 mg l-1). Recent macrolide antibiotics (n=152 children, 28 %) were associated with significantly reduced M. catarrhalis carriage and lower airway infection episodes compared to children who did not receive macrolides; odds ratios 0.19 (95  % CI 0.10-0.35) and 0.15 (0.04-0.41), respectively. CONCLUSION: Despite the frequent use of macrolides, few macrolide-resistant isolates were detected. This suggests a fitness cost associated with macrolide resistance in M. catarrhalis. Macrolide antibiotics remain an effective choice for treating M. catarrhalis lower airway infection in children with chronic endobronchial suppuration.

Nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for clinical microbiology in 2014: General view of the pathogens' antibacterial susceptibility.

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from the patients in Japan was conducted by Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology in 2014. The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period between January 2014 and April 2015 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical Laboratory Standards Institute. Susceptibility testing was evaluated in 1534 strains (335 Staphylococcus aureus, 264 Streptococcus pneumoniae, 29 Streptococcus pyogenes, 281 Haemophilus influenzae, 164 Moraxella catarrhalis, 207 Klebsiella pneumoniae, and 254 Pseudomonas aeruginosa). Ratio of methicillin-resistant S. aureus was 43.6%, and those of penicillin-susceptible S. pneumoniae was 100%. Among H. influenzae, 8.2% of them were found to be ß-lactamase-producing ampicillin-resistant strains, and 49.1% to be ß-lactamase-non-producing ampicillin-resistant strains. Extended spectrum ß-lactamase-producing K. pneumoniae and multi-drug resistant P. aeruginosa with metallo ß-lactamase were 9.2% and 0.4%, respectively.

Efficacy of Aminomethyl Spectinomycins against Complex Upper Respiratory Tract Bacterial Infections.

The most frequent ailment for which antibiotics are prescribed is otitis media (ear infections), which is most commonly caused by Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae Treatment of otitis media is complicated by the fact that the bacteria in the middle ear typically form biofilms, which can be recalcitrant to antibiotic treatment. Furthermore, bacterial respiratory infections can be greatly exacerbated by viral coinfection, which is particularly evidenced by the synergy between influenza and S. pneumoniae In this study, we sought to ascertain the in vivo efficacy of aminomethyl spectinomycin lead 1950, an effective antibacterial agent both in vitro and in vivo against Streptococcus pneumoniae in the context of complex respiratory infections and acute otitis media. A single dose of 1950 significantly reduced bacterial burden in the respiratory tract for all three pathogens, even when species were present in a coinfection model. Additionally, a single dose of 1950 effectively reduced pneumococcal acute otitis media from the middle ear. The agent 1950 also proved efficacious in the context of influenza-pneumococcal super infection. These data further support the development of this family of compounds as potential therapeutic agents against the common causes of complex upper respiratory tract infections and acute otitis media.

Surveillance on susceptibility of strains isolated from pediatric infections.

During the period from January to December 2015, 104 Streptococcus pneumoniae strains, 129 Haemophilus influenzae strains and 54 Moraxella catarrhalis strains isolated from clinical specimens of pediatric infections in the national 16 institutions, studied susceptibilities of total 28 antibiotics, the capsular serotype for S. pneumoniae, the capsular b type and ß-lactamase production capability for H. influenzae, and the ß-lactamase production capability for M. catarrhalis were measured. In S. pneumoniae, the results showed that 68 strains (65.4%) were PSSP, 32 (30.8%) were PISP, and 4 (3.8%) were PRSP. The susceptibilities of TBPM and GRNX among oral antibiotics, and PAPM among injectable antibiotics demonstrated the lowest value with MIC90 ≤ 0.06 µg/mL. The most frequent distribution of S. pneumoniae serotypes was seen in 15B, followed by 19A, and 35B. Serotype strains contained in 13-valent pneumococcal conjugate vaccine (PCV13) were 19 strains (18.3%). In H. influenzae, the results showed that BLNAS accounted for 40 strains (31.0%), BLNAI for 28 strains (21.7%), BLNAR for 47 strains (36.4%), ß-lactamase producing for 14 strains (10.8%). The susceptibilities of quinolones demonstrated the lowest outcome among oral antibiotics with MIC90 ≤ 0.06 µg/mL, and CTRX and TAZ/PIPC (TAZ4 fixed) among injectable antibiotics with MIC of 0.25 µg/mL. There was no detection of capsular type b strains. In M. catarrhalis, all the isolates were ß-lactamase producing strains. The susceptibilities of TBPM, CPFX, TFLX and GRNX among oral antibiotics, and TAZ/PIPC (TAZ4 fixed), PAPM, MEPM and DRPM among injectable antibiotics demonstrated the lowest outcome with MIC of ≤0.06 µg/mL.

Antibacterial Activity of Lefamulin against Pathogens Most Commonly Causing Community-Acquired Bacterial Pneumonia: SENTRY Antimicrobial Surveillance Program (2015-2016).

Lefamulin, the first semisynthetic pleuromutilin antibacterial for intravenous and oral treatment of community-acquired bacterial pneumonia (CABP), and comparators were evaluated for in vitro activity against a global collection of pathogens commonly causing CABP (n = 8595) from the 2015 and 2016 SENTRY Antimicrobial Surveillance Program. Lefamulin was highly active against the pathogens Streptococcus pneumoniae, including multidrug-resistant and extensively drug-resistant strains (MIC50/90 for total and resistant subsets, 0.06/0.12 µg/ml; 100% inhibited at ≤1 µg/ml), Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA; both MIC50/90, 0.06/0.12 µg/ml; 99.8% and 99.6% inhibited at ≤1 µg/ml, respectively), Haemophilus influenzae (MIC50/90, 0.5/1 µg/ml; 93.8% inhibited at ≤1 µg/ml), and Moraxella catarrhalis (MIC50/90, 0.06/0.12 µg/ml; 100% inhibited at ≤0.25 µg/ml), and its activity was unaffected by resistance to other antibacterial classes.

Antimicrobial susceptibility of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis clinical isolates from children with acute otitis media in Japan from 2014 to 2017.

Increase in antimicrobial resistance (AMR) among pathogenic bacteria is a serious threat to public health. Surveillance studies to monitor shifting trends in resistance are important and guide the selection of appropriate antimicrobial agents for a particular organism. Furthermore, these studies help in dissemination of accurate information regarding AMR to the public. In this study, we investigated the antimicrobial susceptibility patterns of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis clinical isolates from outpatient children with acute otitis media in Japan from 2014 to 2017. A total of 8693 strains (2415 of S. pneumoniae, 3657 of H. influenzae, and 2621 of M. catarrhalis) were clinically isolated, and their antimicrobial susceptibilities to benzylpenicillin (PCG), ampicillin (ABPC), amoxicillin-clavulanic (AMPC/CVA), azithromycin (AZM), ceftriaxone (CTRX), and levofloxacin (LVFX) were investigated. Based on the minimum inhibitory concentration (MIC) breakpoints, the average proportion of S. pneumoniae isolates non-susceptible to PCG and AZM was 38.2% and 82.0% respectively. The average proportion of H. influenzae isolates non-susceptible to ABPC, CVA/AMPC, and CTRX was 61.9%, 43.5%, and 49.4%, respectively. The high prevalence of these resistant organisms is attributed to frequent use of antibiotic agents in Japan. Moreover, the proportion of LVFX-non-susceptible H. influenzae isolates increased in this four-year study. Here, we report updates regarding the AMR trends amongst the major pathogens that cause acute otitis media in Japan. Continuing surveillance of antimicrobial susceptibility and application of control measures against further transmission are required to decrease the emergence of resistant strains.

Antibodies against the Majority Subunit (PilA) of the Type IV Pilus of Nontypeable Haemophilus influenzae Disperse Moraxella catarrhalis from a Dual-Species Biofilm.

Otitis media (OM) is often polymicrobial, with nontypeable Haemophilus influenzae (NTHI) and Moraxella catarrhalis (Mcat) frequently cocultured from clinical specimens. Bacterial biofilms in the middle ear contribute to the chronicity and recurrence of OM; therefore, strategies to disrupt biofilms are needed. We have focused our vaccine development efforts on the majority subunit of NTHI type IV pili, PilA. Antibodies against a recombinant, soluble form of PilA (rsPilA) both disrupt and prevent the formation of NTHI biofilms in vitro. Moreover, immunization with rsPilA prevents and resolves NTHI-induced experimental OM. Here, we show that antibodies against rsPilA also prevent and disrupt polymicrobial biofilms. Dual-species biofilms formed by NTHI and Mcat at temperatures that mimic the human nasopharynx (34°C) or middle ear (37°C) were exposed to antiserum against either rsPilA or the OMP P5 adhesin of NTHI. NTHI+Mcat biofilm formation was significantly inhibited by antiserum directed against both adhesin proteins at either temperature. However, only anti-rsPilA disrupted NTHI+Mcat preestablished biofilms at either temperature and actively dispersed both NTHI and Mcat via interspecies quorum signaling. Newly released NTHI and Mcat were significantly more susceptible to killing by antibiotics. Taken together, these results revealed new opportunities for treatment of biofilm-associated diseases via a strategy that combines vaccine-induced antibody-mediated biofilm dispersal with traditional antibiotics, at a significantly reduced dosage to exploit the newly released, antibiotic-sensitive phenotype. Combined, our data strongly support the utility of rsPilA both as a preventative and as a therapeutic vaccine antigen for polymicrobial OM due to NTHI and Mcat.IMPORTANCE Middle ear infections (or otitis media [OM]) are highly prevalent among children worldwide and present a tremendous socioeconomic challenge for health care systems. More importantly, this disease diminishes the quality of life of young children. OM is often chronic and recurrent, due to the presence of highly antibiotic-resistant communities of bacteria (called biofilms) that persist within the middle ear space. To combat these recalcitrant infections, new and powerful biofilm-directed approaches are needed. Here, we describe the ability to disrupt a biofilm formed by the two most common bacteria that cause chronic and recurrent OM in children, via an approach that combines the power of vaccines with that of traditional antibiotics. An outcome of this strategy is that antibiotics can more easily kill the bacteria that our vaccine-induced antibodies have released from the biofilm. We believe that this approach holds great promise for both the prevention and treatment of OM.

Antimicrobial activity of ceftaroline and comparator agents tested against organisms isolated from patients with community-acquired bacterial pneumonia in Europe, Asia, and Latin America.

OBJECTIVE: To evaluate the potency and spectrum of ceftaroline and comparator agents tested against contemporary bacteria isolated from patients with community-acquired bacterial pneumonia (CABP) in Europe (EUR), Asia-Pacific (APAC), and Latin America (LATAM). METHODS: A total of 4321 bacterial isolates were collected consecutively by the SENTRY Antimicrobial Surveillance Program in 2015-2017 from 65 medical centers located in Western Europe (W-EUR; 21 centers in 10 nations), Eastern Europe and the Mediterranean region (E-EUR; 15 centers in 11 nations), APAC (18 centers in nine nations), and LATAM (11 centers in nine nations). Isolates were collected from lower respiratory tract specimens, and an isolate obtained from an outpatient or earlier than 48h after hospitalization was considered community-acquired. Organisms were tested for susceptibility by reference broth microdilution methods in a central laboratory. RESULTS: Among Streptococcus pneumoniae (n=1736), 99.9% of isolates were ceftaroline-susceptible (MIC50/90, 0.008/0.12mg/l), and ceftriaxone susceptibility (≤1mg/l) ranged from 97.4% in W-EUR to 85.3% in the APAC region. Ceftaroline was also active against Haemophilus influenzae (n=1172; MIC50/90, 0.008/0.03mg/l; 99.8%/93.9% susceptible per CLSI/EUCAST criteria) and Staphylococcus aureus (n=777; MIC50/90, 0.25/1mg/l; 97.4% susceptible). Oxacillin resistance ranged from 31.9% in the APAC region to 15.0% in E-EUR. Ceftaroline also demonstrated potent activity against Moraxella catarrhalis (n=613; MIC50/90, 0.06/0.25mg/l) and Haemophilus parainfluenzae (n=23; MIC50/90, 0.015/0.03mg/l). CONCLUSIONS: Susceptibility rates varied widely by geographic region. Ceftaroline was active against the vast majority of bacterial organisms isolated from patients with CABP.

Biochemical and biophysical characterization of 1,4-naphthoquinone as a dual inhibitor of two key enzymes of type II fatty acid biosynthesis from Moraxella catarrhalis.

The fatty acid biosynthesis (FAS II) is a vital process in bacteria and regarded as an attractive pathway for the development of potential antimicrobial agents. In this study, we report 1,4-naphthoquinone (NPQ) as a dual inhibitor of two key enzymes of FAS II pathway, namely FabD (Malonyl-CoA:ACP transacylase) and FabZ (ß-hydroxyacyl-ACP dehydratase). Mode of inhibition of NPQ was found to be non-competitive for both enzymes with IC50 of 26.67 µΜ and 23.18 µΜ against McFabZ and McFabD respectively. Conformational changes in secondary and tertiary structures marked by the loss of helical contents were observed in both enzymes upon NPQ binding. The fluorescence quenching was found to be static with a stable ground state complex formation. ITC based studies have shown that NPQ is binding to McFabZ with a stronger affinity (~1.5×) as compared to McFabD. Molecular docking studies have found that NPQ interacts with key residues of both McFabD (Ser209, Arg126, and Leu102) and McFabZ (His74 and Tyr112) enzymes. Both complexes have shown the structural stability during the 20 ns run of molecular dynamics based simulations. Altogether, the present study suggests that NPQ scaffold can be exploited as a multi-targeted inhibitor of FAS II pathway, and these biochemical and biophysical findings will further help in the development of potent antibacterial agents targeting FAS II pathway.