Molecular Characterization of Streptococcus pyogenes Causing Invasive Disease in Pediatric Population in Spain A 12-year Study.

OBJECTIVES: To perform a comprehensive description of the epidemiology of Streptococcus pyogenes invasive disease in the pediatric population in 2 regions of Spain (Catalonia and Gipuzkoa) through 12 years. METHODS: All S. pyogenes isolates causing invasive disease in pediatric patients between 2005 and 2016 were included. The emm-type and the presence of 13 exotoxin genes (speA, speB, speC, speF, speG, speH, speI, speJ, speK, speL, speM, smeZ, ssa and slo) were determined in all 93 available isolates and the Multi Locus Sequece Typing in 10% of isolates of each different emm-type. RESULTS: Overall, 103 cases of S. pyogenes invasive infections were detected: 77 in Catalonia and 26 in Gipuzkoa, being 50.5% females. The incidence rate per 100,000 children was 2.5 for Gipuzkoa and 2.6 for Catalonia, with no significant temporal trends. The median age was 30 months. The most frequent clinical presentations were: pneumonia (26.2%), bacteremia/sepsis (23.3%), septic arthritis/osteomyelitis (22.3%), cellulitis/mastoiditis (12.6%) and meningitis (6.8%). Eight children developed streptococcal toxic shock syndrome. Nine cases were preceded by varicella infection. The associated mortality rate was 3.9%. Three isolates were resistant to erythromycin, being one of them also resistant to clindamycin and 4 isolates were resistant to levofloxacine. Forteen different emm-types were detected being emm1/ST28 (40.9%) the most frequent clone in both regions followed by emm12/ST36-ST242, emm6/ST382, emm3/ST15, emm75/ST150 and emm4/ST38-39. speA gene was only detected in emm1 and emm3 isolates. Eight exotoxins were enough to assign an emm-type with a very high degree of accuracy (95%). The 30-valent vaccine would include 96.8% of isolates.

Changes in the serotype distribution of Streptococcus pneumoniae causing otitis media after PCV13 introduction in Spain.

One of the beneficial effects of pneumococcal conjugate vaccines (PCVs) has been a decrease in the incidence of non-invasive infections, such as otitis media (OM) caused by vaccine serotypes. In this study, we analyzed the epidemiology of pneumococcal OM before and after PCV13 introduction in 2010. Between 2008 and 2016, the middle ear exudates from 2653 children under 14 years of age with OM were studied in two Spanish provinces (Gipuzkoa and Barcelona), and S. pneumoniae was isolated in 235 (8.9%) of cases. The 204 available isolates were serotyped and distributed in three 3-year periods: one before and two after PCV13 introduction (early and late post-PCV13). A significant decrease in the rate of OM caused by S. pneumoniae was observed mainly due to a decrease in infections caused by all PCV13 serotypes, although exceptions were observed including the persistence of serotype 3 in Gipuzkoa and a weak re-emergence of serotype 19F in both regions. The rate and diversity of non-PCV13 serotypes increased in both regions and an emerging clone causing OM was detected in each region: serotype 23B ST2372 in Gipuzkoa and serotype 11A ST838/ST6521 in Barcelona. The introduction of PCV13 has been followed by a change in the epidemiology of pneumococcal OM, with a decrease in the rate of vaccine serotypes accompanied by an increase in the diversity of non-vaccine serotype and the clonal spreading of different single clones in each region.

Synergistic Activity of Niclosamide in Combination With Colistin Against Colistin-Susceptible and Colistin-Resistant Acinetobacter baumannii and Klebsiella pneumoniae.

Colistin is among the few antibiotics effective against multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae clinical isolates. However, in the last few years, colistin-resistant A. baumannii and K. pneumoniae strains have emerged. Therefore, combination therapies, between colistin and other old drugs, restoring the activity of colistin are required. The main objective of this study was to analyse the activity of niclosamide, an anthelmintic drug, in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) A. baumannii and K. pneumoniae. The MIC were determined by microdilution assay and the time-kill curves were performed. The zeta potential of Col-S and Col-R of A. baumannii and K. pneumoniae in presence of niclosamide was assessed. Niclosamide in combination with colistin showed improved activity against Col-S and Col-R A. baumannii and K. pneumoniae. Time-killing curves showed synergic activity between niclosamide and colistin against Col-S and Col-R A. baumannii and K. pneumoniae, especially when niclosamide or colistin was added for second time at 4 h of the 24 h killing curve. Col-R A. baumannii and K. pneumoniae in presence of niclosamide exhibited a greater negative charge (-34.95 ± 0.35 mV and -38.85 ± 0.92 mV; P < 0.05) than Col-R A. baumannii and K. pneumoniae in absence of niclosamide (-26.85 ± 3.65 mV and -35.27 ± 0.72 mV). These data suggest that niclosamide might be combined with colistin, being a potential alternative for treatment of Col-R Gram-negative bacilli infections.

Synergistic activity of an OmpA inhibitor and colistin against colistin-resistant Acinetobacter baumannii: mechanistic analysis and in vivo efficacy.

Objectives: Preventing bacterial contact with host cells can provide an additional approach to tackling MDR Acinetobacter baumannii. Recently, we identified AOA-2 as a potential blocker of A. baumannii outer membrane protein A without presenting bactericidal activity. Here, we aimed to study whether AOA-2 can increase the activity of colistin against colistin-resistant A. baumannii in vitro and in vivo. Methods: Reference and clinical A. baumannii strains susceptible and resistant to colistin (CST-S and CST-R) were used. Microdilution and time-kill curve assays were performed to determine the synergy between AOA-2 and colistin. SDS-PAGE assays with CST-S and CST-R outer membrane proteins and MALDI-TOF-TOF (MS-MS/MS) analysis were performed to determine the AOA-2 and colistin synergy mechanism. In a murine peritoneal sepsis model, the therapeutic efficacy of AOA-2 (10 mg/kg/24 h) in combination with a sub-optimal dose of colistin (10 mg/kg/24 h) against CST-R was evaluated by determining the bacterial load in tissues and blood, and mouse survival. Results: We showed that AOA-2 increased the in vitro colistin susceptibility of reference and clinical CST-S and CST-R strains. This combination also enhanced their killing activity after 24 h of drug exposure. This synergy is mediated by the overexpression of Omp25. In vivo, the combination of AOA-2 with colistin significantly reduced the bacterial load in tissues and blood, and increased mouse survival, compared with colistin monotherapy. Conclusions: We identified a novel class of antimicrobial agents that has proven to be effective in combination with colistin in an experimental model of severe infection by CST-R A. baumannii.

Combating virulence of Gram-negative bacilli by OmpA inhibition.

Preventing the adhesion of pathogens to host cells provides an innovative approach to tackling multidrug-resistant bacteria. In this regard, the identification of outer membrane protein A (OmpA) as a key bacterial virulence factor has been a major breakthrough. The use of virtual screening helped us to identify a cyclic hexapeptide AOA-2 that inhibits the adhesion of Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli to host cells and the formation of biofilm, thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. Inhibition of OmpA offers a strategy as monotherapy to address the urgent need for treatments for infections caused by Gram-negative bacilli.

Overproduction of Outer Membrane Protein A by Acinetobacter baumannii as a Risk Factor for Nosocomial Pneumonia, Bacteremia, and Mortality Rate Increase.

Background: Outer membrane protein A (OmpA) is a porin involved in Acinetobacter baumannii pathogenesis. However, OmpA clinical implication in hospital-acquired infections remains unknown. We aimed to determine whether OmpA overproduction was a risk factor associated with pneumonia, bacteremia, and mortality. Methods: We analyzed demographic, microbiological, and clinical data from 100 patients included in a unicenter cohort and 246 included in a unicenter cohort and a multicenter cohort. Representative isolates were classified into 2 groups: (1) isolates from patients colonized by A. baumannii (16 from the unicenter and 20 from the multicenter cohort) and (2) isolates from bacteremic or nonbacteremic patients with pneumonia (PP) caused by A. baumannii (13 from the unicenter and 23 from the multicenter cohort) Expression of ompA was determined with quantitative reverse-transcription polymerase chain reaction. Results: Isolates from PP overexpressed more ompA than those from colonized patients from the unicenter (ratio, 1.76 vs 0.36; P < .001) and the multicenter (1.36 vs 0.91; P = .03) cohorts. Among isolates from PP, those from bacteremic patients overexpressed nonsignificantly more ompA than those from nonbacteremic patients in the unicenter (ratio, 2.37 vs 1.43; P = .06) and the multicenter (2.03 vs 0.91; P = .14) cohorts. Multivariate analysis in both cohorts together showed ompA overexpression as independent risk factor for pneumonia (P < .001), bacteremia (P = .005), and death (P = .049). Conclusions: These data suggest that ompA overexpression is an associated factor for pneumonia, bacteremia, and death due to A. baumannii.

Targeted delivery of HGF to the skeletal muscle improves glucose homeostasis in diet-induced obese mice

Hepatocyte growth factor (HGF) is a cytokine that increases glucose transport ex vivo in skeletal muscle. The aim of this work was to decipher the impact of whether conditional overexpression of HGF in vivo could improve glucose homeostasis and insulin sensitivity in mouse skeletal muscle. Following tetracyclin administration, muscle HGF levels were augmented threefold in transgenic mice (SK-HGF) compared to control mice without altering plasma HGF levels. In conditions of normal diet, SK-HGF mice showed no differences in body weight, plasma triglycerides, blood glucose, plasma insulin and glucose tolerance compared to control mice. Importantly, obese SK-HGF mice exhibited improved whole-body glucose tolerance independently of changes in body weight or plasma triglyceride levels compared to control mice. This effect on glucose homeostasis was associated with significantly higher (∼80 %) levels of phosphorylated protein kinase B in muscles from SK-HGF mice compared to control mice. In conclusion, muscle expression of HGF counteracts obesity-mediated muscle insulin resistance and improves glucose tolerance in mice

Targeted delivery of HGF to the skeletal muscle improves glucose homeostasis in diet-induced obese mice.

Hepatocyte growth factor (HGF) is a cytokine that increases glucose transport ex vivo in skeletal muscle. The aim of this work was to decipher the impact of whether conditional overexpression of HGF in vivo could improve glucose homeostasis and insulin sensitivity in mouse skeletal muscle. Following tetracyclin administration, muscle HGF levels were augmented threefold in transgenic mice (SK-HGF) compared to control mice without altering plasma HGF levels. In conditions of normal diet, SK-HGF mice showed no differences in body weight, plasma triglycerides, blood glucose, plasma insulin and glucose tolerance compared to control mice. Importantly, obese SK-HGF mice exhibited improved whole-body glucose tolerance independently of changes in body weight or plasma triglyceride levels compared to control mice. This effect on glucose homeostasis was associated with significantly higher (∼80%) levels of phosphorylated protein kinase B in muscles from SK-HGF mice compared to control mice. In conclusion, muscle expression of HGF counteracts obesity-mediated muscle insulin resistance and improves glucose tolerance in mice.

Role of OmpA in the multidrug resistance phenotype of Acinetobacter baumannii.

Acinetobacter baumannii has emerged as a nosocomial pathogen with an increased prevalence of multidrug-resistant strains. The role of the outer membrane protein A (OmpA) in antimicrobial resistance remains poorly understood. In this report, disruption of the ompA gene led to decreased MICs of chloramphenicol, aztreonam, and nalidixic acid. We have characterized, for the first time, the contribution of OmpA in the antimicrobial resistance phenotype of A. baumannii.