The small quinolone derived compound HT61 enhances the effect of tobramycin against Pseudomonas aeruginosa in vitro and in vivo.

HT61 is a small quinolone-derived compound previously demonstrated to exhibit bactericidal activity against gram-positive bacteria including methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). When combined with the classical antibiotics and antiseptics neomycin, gentamicin, mupirocin and chlorhexidine, HT61 demonstrated synergistic bactericidal activity against both MSSA and MRSA infections in vitro. In this study, we investigated the individual antimicrobial activity of HT61 alongside its capability to increase the efficacy of tobramycin against both a tobramycin sensitive laboratory reference strain (PAO1) and tobramycin resistant clinical isolates (RP73, NN2) of the gram-negative bacteria Pseudomonas aeruginosa (P. aeruginosa). Using broth microdilution methods, the MICs of HT61 against all strains were assessed, as well as the effect of HT61 in combination with tobramycin using both the chequerboard method and bacterial time-kill assays. A murine model of pulmonary infection was also used to evaluate the combination therapy of tobramycin and HT61 in vivo. In these studies, we demonstrated significant synergism between HT61 and Tobramycin against the tobramycin resistant P. aeruginosa strains RP73 and NN2, whilst an additive/intermediate effect was observed for P. aeruginosa strain PA01 which was further confirmed using bacterial time kill analysis. In addition, the enhancement of tobramycin by HT61 was also evident in in vitro assays of biofilm eradication. Finally, in vivo studies revealed analogous effects to those observed in vitro with HT61 when administered in combination with tobramycin against each of the three P. aeruginosa strains at the highest tested dose (10 mg/kg)

In vitro activity of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole against planktonic and sessile Burkholderia cepacia complex bacteria

Objectives: The goal of the present study was to obtain a comprehensive overview of the bacteriostatic and bactericidal effects of six commonly used antibiotics on planktonic as well as on sessile Burkholderia cepacia complex cells. Methods: The bacteriostatic and bactericidal activities of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole were determined against 38 B. cepacia complex strains. MICs and minimal biofilm inhibitory concentrations (MBICs) were determined using a traditional broth microdilution method and a novel resazurin-based viability staining, respectively. The bactericidal effects of the investigated antibiotics (using antibiotic concentrations corresponding to 10 x MIC; except for tobramycin, for which a final concentration of 4 x MIC was tested) on stationary phase planktonic cultures and on 24-h-old biofilms were evaluated using conventional plate count methods. Results: Our results confirm the innate resistance of B. cepacia complex organisms to six first-line antibiotics used to treat infected cystic fibrosis patients. All antibiotics showed similar bacteriostatic activities against exponentially growing B. cepacia complex planktonic cells and freshly adhered sessile cells (4 h). In addition, most of the antibiotics showed similar bactericidal effects on stationary phase planktonic cultures and on young and older biofilms. Conclusions: Despite the general assumption that sessile cells show a decreased susceptibility to antibiotics, our data indicate similar bacteriostatic and bactericidal activity of six selected antibiotics against planktonic and sessile B. cepacia complex bacteria

Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems

Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics

<div><p>Antibiotic susceptibility of bacterial pathogens is typically evaluated using <i>in vitro</i> assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy <i>in vitro</i> and <i>in vivo</i>, with some antibiotics being effective <i>in vitro</i> but not <i>in vivo</i> or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen <i>Pseudomonas aeruginosa</i>, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an <i>in vivo</i>-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against <i>P</i>. <i>aeruginosa</i>, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics <i>in vitro</i> and <i>in vivo</i> may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity.</p></div

Pseudomonas aeruginosa bacteremia in patients undergoing liver transplantation: An emerging problem

In our institution, Pseudomonas aeruginosa bacteremia appeared to occur with increasing frequency in patients undergoing liver transplantation. We thus conducted a prospective study to define risk factors and outcome in these patients. Over a 19-month period 6% of liver transplants were followed by Pseudomonas bacteremia. The mean age was 46 years (range, 24 to 67 years). The interval between transplantation and onset of bacteremia was 3 to 372 days (mean, 80). The incidence of Pseudomonas bacteremia in liver transplants was three times that of other transplants (heart, lung, kidney). Ninety one percent of infections were nosocomial. Polymicrobial bacteremia occurred in 30% of episodes. The portal of entry was respiratory in 30%, abdominal in 35%, and biliary in 13%. Four patients had recurrent Pseudomonas bacteremia: liver abscess (1), biliary obstruction (2), subhepatic abscess (1). Survival at 14 days was 70%. Survival rates were significantly lower for patients with hypotension, on mechanical ventilators, and increasing severity of illness (p < 0.05). Survival was higher when bacteremia occurred within the first 30 days after transplantation compared to after 30 days. A large number (43.4%) of Pseudomonas bacteremias occurred after transplant surgery or biliary tract manipulation, while the patient was receiving a prophylactic regimen of cefotaxime and ampicillin. P. aeruginosa is an important pathogen in the liver transplant recipient; prevention may be possible for a subgroup of patients with the use of prophylactic antibiotics with activity against P. aeruginosa

Antimicrobial efficacy against Pseudomonas aeruginosa biofilm formation in a three-dimensional lung epithelial model and the influence of fetal bovine serum

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS

Results of A Local Combination Therapy Antibiogram For \u3cem\u3ePseudomonas Aeruginosa\u3c/em\u3e Isolates: Is Double Worth The Trouble?

Purpose: To determine the frequency at which fluoroquinolones and aminoglycosides demonstrate in vitro activity against non-urinary, non-skin/skin structure Pseudomonas aeruginosa isolates exhibiting decreased susceptibilities to one or more β-lactam agents. Methods: β-lactam-non-susceptible P. aeruginosa isolates recovered from blood, bone, lower respiratory tract, pleural fluid, cerebrospinal fluid, or peritoneal fluid cultures between October 2010 and October 2014 were reviewed from four community hospitals within a single health-system. Only the first isolate per patient was included for analysis. The likelihood that each isolate was susceptible to a non-β-lactam antimicrobial was then determined and summarized within a combination antibiogram. Results: In total, 179 P. aeruginosa isolates with decreased susceptibilities to one or more β-lactam agents were assessed. Because no appreciable differences in antimicrobial susceptibility profile were observed between hospitals, the isolates were evaluated in aggregate. Susceptibility rates for β-lactam monotherapy ranged from 34% to 75%. Aminoglycosides possessed increased antibacterial activity compared to fluoroquinolones. Tobramycin was the non-β-lactam most likely to expand antimicrobial coverage against β-lactam-non-susceptible P. aeruginosa with activity against 64%, 66%, and 65% of cefepime-, piperacillin-tazobactam-, and meropenem-non-susceptible isolates, respectively (p \u3c 0.001 for all). Conclusions: The results of this study support the use of aminoglycosides over fluoroquinolones for achieving optimal, empiric antimicrobial combination therapy for P. aeruginosa when dual antimicrobial therapy is clinically necessary. Future efforts aimed at optimizing combination therapy for P. aeruginosa should focus on systemic interventions that limit the selection of fluoroquinolones in combination with β-lactams to expand coverage based on local susceptibility rates

Developing selective media for quantification of multispecies biofilms following antibiotic treatment

The lungs of cystic fibrosis (CF) patients are chronically colonized by a polymicrobial biofilm community, leading to difficult-to-treat infections. To combat these infections, CF patients are commonly treated with a variety of antibiotics. Understanding the dynamics of polymicrobial community composition in response to antibiotic therapy is essential in the search for novel therapies. Culture-dependent quantification of individual bacteria from defined multi-species biofilms is frequently carried out by plating on selective media. However, the influence of the selective agents in these media on quantitative recovery before or after antibiotic treatment is often unknown. In the present study we developed selective media for six bacterial species that are frequently co-isolated from the CF lung, i.e. Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans. We show that certain supplementations to selective media strongly influence quantitative recovery of (un) treated biofilms. Hence, the developed media were optimized for selectivity and quantitative recovery before or after treatment with antibiotics of four major classes, i.e. ceftazidime, ciprofloxacin, colistin, or tobramycin. Finally, in a proof of concept experiment the novel selective media were applied to determine the community composition of multispecies biofilms before and after treatment with tobramycin