D-amino acids enhance the activity of antimicrobials against biofilms of clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa.
ABSTRACT: Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of D-amino acids (D-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of D-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. D-Met, D-Phe, and D-Trp at concentrations of ? 5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (D-Met/D-Phe/D-Trp). When combined with D-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 ?g/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of D-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 ?g/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of D-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity.
Project description:Both Staphylococcus aureus and Staphylococcus epidermidis are commonly associated with periprosthetic joint infections (PJIs). The treatment of PJI can be challenging because biofilms are assumed to have an increased intolerance to antibiotics. This makes the treatment of PJI challenging from a clinical perspective. Although S. aureus has been previously demonstrated to have increased biofilm antibiotic tolerance, this has not been well established with Staphylococcus epidermidis. A prospective registry of PJI S. epidermidis isolates was developed. The efficacy of clinically relevant antibiotics was quantified against these isolates. S. epidermidis planktonic minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were collected using clinical laboratory standard index (CLSI) assays for eight antibiotics (doxycycline, vancomycin, daptomycin, clindamycin, rifampin, nafcillin, and trimethoprim/sulfamethoxazole). Mature biofilms were grown in vitro, after which minimum biofilm inhibitory concentration (MBIC) and minimum biofilm bactericidal concentration (MBBC) were quantified. Only rifampin and doxycycline had a measurable MBIC across all tested isolates. Based on MBBC, 64% of S. epidermidis biofilms could be eliminated by rifampin, whereas only 18% by doxycycline. S. epidermidis biofilm was observed to have a high tolerance to antibiotics as compared to planktonic culture. Isolate biofilm antibiotic tolerance varied to a larger degree than was seen in planktonic cultures.
Project description:Many Pseudomonas aeruginosa isolates from the airways of patients with cystic fibrosis (CF) are sensitive to antibiotics in susceptibility testing, but eradication of the infection is difficult. The main reason is the biofilm formation in the airways of patients with CF. The pharmacokinetics (PKs) and pharmacodynamics (PDs) of antimicrobials can reliably be used to predict whether antimicrobial regimens will achieve the maximum bactericidal effect against infections. Unfortunately, however, most PK/PD studies of antimicrobials have been done on planktonic cells and very few PK/PD studies have been done on biofilms, partly due to the lack of suitable models in vivo. In the present study, a biofilm lung infection model was developed to provide an objective and quantitative evaluation of the PK/PD profile of antimicrobials. Killing curves were set up to detect the antimicrobial kinetics on planktonic and biofilm P. aeruginosa cells in vivo. Colistin showed concentration-dependent killing, while imipenem showed time-dependent killing on both planktonic and biofilm P. aeruginosa cells in vivo. The parameter best correlated to the elimination of bacteria in lung by colistin was the area under the curve (AUC) versus MIC (AUC/MIC) for planktonic cells or the AUC versus minimal biofilm inhibitory concentration (MBIC; AUC/MBIC) for biofilm cells. The best-correlated parameter for imipenem was the time that the drug concentration was above the MIC for planktonic cells (T(MIC)) or time that the drug concentration was above the MBIC (T(MBIC)) for biofilm cells. However, the AUC/MIC of imipenem showed a better correlation with the efficacy of imipenem for biofilm infections (R(2) = 0.89) than planktonic cell infections (R(2) = 0.38). The postantibiotic effect (PAE) of colistin and imipenem was shorter in biofilm infections than planktonic cell infections in this model.
Project description:P128 is an antistaphylococcal protein, comprising a cell wall-degrading enzymatic region and a Staphylococcus-specific binding region, which possesses specific and potent bactericidal activity against sensitive and drug-resistant strains of Staphylococcus aureus To explore P128's ability to kill S. aureus in a range of environments relevant to clinical infection, we investigated the anti-S. aureus activity of P128 alone and in combination with standard-of-care antibiotics on planktonic and biofilm-embedded cells. P128 was found to have potent antibiofilm activity on preformed S. aureus biofilms as detected by CFU reduction and a colorimetric minimum biofilm inhibitory concentration (MBIC) assay. Scanning electron microscopic images of biofilms formed on the surfaces of microtiter plates and on catheters showed that P128 at low concentrations could destroy the biofilm structure and lyse the cells. When it was tested in combination with antibiotics which are known to be poor inhibitors of S. aureus in biofilms, such as vancomycin, gentamicin, ciprofloxacin, linezolid, and daptomycin, P128 showed highly synergistic antibiofilm activity that resulted in much reduced MBIC values for P128 and the individual antibiotics. The synergistic effect was seen for both sensitive and resistant isolates of S. aureus Additionally, in an in vitro mixed-biofilm model mimicking the wound infection environment, P128 was able to prevent biofilm formation by virtue of its anti-Staphylococcus activity. The potent S. aureus biofilm-inhibiting activity of P128 both alone and in combination with antibiotics is an encouraging sign for the development of P128 for treatment of complicated S. aureus infections involving biofilms.
Project description:Resistance to ?-lactam antibiotics is a frequent problem in Pseudomonas aeruginosa lung infection of cystic fibrosis (CF) patients. This resistance is mainly due to the hyperproduction of chromosomally encoded ?-lactamase and biofilm formation. The purpose of this study was to investigate the role of ?-lactamase in the pharmacokinetics (PK) and pharmacodynamics (PD) of ceftazidime and imipenem on P. aeruginosa biofilms. P. aeruginosa PAO1 and its corresponding ?-lactamase-overproducing mutant, PA?DDh2Dh3, were used in this study. Biofilms of these two strains in flow chambers, microtiter plates, and on alginate beads were treated with different concentrations of ceftazidime and imipenem. The kinetics of antibiotics on the biofilms was investigated in vitro by time-kill methods. Time-dependent killing of ceftazidime was observed in PAO1 biofilms, but concentration-dependent killing activity of ceftazidime was observed for ?-lactamase-overproducing biofilms of P. aeruginosa in all three models. Ceftazidime showed time-dependent killing on planktonic PAO1 and PA?DDh2Dh3. This difference is probably due to the special distribution and accumulation in the biofilm matrix of ?-lactamase, which can hydrolyze the ?-lactam antibiotics. The PK/PD indices of the AUC/MBIC and C(max)/MBIC (AUC is the area under concentration-time curve, MBIC is the minimal biofilm-inhibitory concentration, and C(max) is the maximum concentration of drug in serum) are probably the best parameters to describe the effect of ceftazidime in ?-lactamase-overproducing P. aeruginosa biofilms. Meanwhile, imipenem showed time-dependent killing on both PAO1 and PA?DDh2Dh3 biofilms. An inoculum effect of ?-lactams was found for both planktonic and biofilm P. aeruginosa cells. The inoculum effect of ceftazidime for the ?-lactamase-overproducing mutant PA?DDh2Dh3 biofilms was more obvious than for PAO1 biofilms, with a requirement of higher antibiotic concentration and a longer period of treatment.
Project description:The time course of activity of colistin and imipenem against mucoid and nonmucoid Pseudomonas aeruginosa growing in a biofilm showed that compared with those for planktonic bacteria, the kinetics of colistin and imipenem retained the concentration- and time-dependent killing, respectively, but higher doses of antibiotics and longer dosing periods were required for biofilm eradication. Biofilms of mucoid P. aeruginosa were more difficult to eradicate than nonmucoid biofilms.
Project description:Tolerance towards antibiotics of Pseudomonas aeruginosa biofilms is recognized as a major cause of therapeutic failure of chronic lung infection in cystic fibrosis (CF) patients. This lung infection is characterized by antibiotic-tolerant biofilms in mucus with zones of O2 depletion mainly due to polymorphonuclear leukocytic activity. In contrast to the main types of bactericidal antibiotics, it has not been possible to establish an association between the bactericidal effects of colistin and the production of detectable levels of OH?? on several strains of planktonic P. aeruginosa. Therefore, we propose that production of OH?? may not contribute significantly to the bactericidal activity of colistin on P. aeruginosa biofilm. Thus, we investigated the effect of colistin treatment on biofilm of wild-type PAO1, a catalase-deficient mutant (?katA) and a colistin-resistant CF isolate cultured in microtiter plates in normoxic- or anoxic atmosphere with 1 mM nitrate. The killing of bacteria during colistin treatment was measured by CFU counts, and the OH? formation was measured by 3(')-(p-hydroxylphenyl fluorescein) fluorescein (HPF) fluorescence. Validation of the assay was done by hydrogen peroxide treatment. OH? formation was undetectable in aerobic PAO1 biofilms during 3 h of colistin treatment. Interestingly, we demonstrate increased susceptibility of P. aeruginosa biofilms towards colistin during anaerobic conditions. In fact, the maximum enhancement of killing by anaerobic conditions exceeded 2 logs using 4 mg L(-1) of colistin compared to killing at aerobic conditions.
Project description:The lungs of cystic fibrosis (CF) patients are often chronically colonized by multiple microbial species that can form biofilms, including the major CF pathogen <i>Pseudomonas aeruginosa</i>. Herewith, lower microbial diversity in CF airways is typically associated with worse health outcomes. In an attempt to treat CF lung infections patients are frequently exposed to antibiotics, which may affect microbial diversity. This study aimed at understanding if common antibiotics that target <i>P. aeruginosa</i> influence microbial diversity. To this end, a microaerophilic multispecies biofilm model of frequently co-isolated members of the CF lung microbiome (<i>Pseudomonas aeruginosa</i>, <i>Staphylococcus aureus</i>, <i>Streptococcus anginosus</i>, <i>Achromobacter xylosoxidans</i>, <i>Rothia mucilaginosa</i>, and <i>Gemella haemolysans</i>) was exposed to antipseudomonal antibiotics. We found that antibiotics that affected several dominant species (i.e. ceftazidime, tobramycin) resulted in higher species evenness compared to colistin, which is only active against <i>P. aeruginosa</i>. Furthermore, susceptibility of individual species in the multispecies biofilm following antibiotic treatment was compared to that of the respective single-species biofilms, showing no differences. Adding three anaerobic species (<i>Prevotella melaninogenica</i>, <i>Veillonella parvula</i>, and <i>Fusobacterium nucleatum</i>) to the multispecies biofilm did not influence antibiotic susceptibility. In conclusion, our study demonstrates antibiotic-dependent effects on microbial community diversity of multispecies biofilms comprised of CF microbiome members.
Project description:Drug resistance and tolerance greatly diminish the therapeutic potential of antibiotics against pathogens. Antibiotic tolerance by bacterial biofilms often leads to persistent infections, but its mechanisms are unclear. Here we use a proteomics approach, pulsed stable isotope labelling with amino acids (pulsed-SILAC), to quantify newly expressed proteins in colistin-tolerant subpopulations of Pseudomonas aeruginosa biofilms (colistin is a 'last-resort' antibiotic against multidrug-resistant Gram-negative pathogens). Migration is essential for the formation of colistin-tolerant biofilm subpopulations, with colistin-tolerant cells using type IV pili to migrate onto the top of the colistin-killed biofilm. The colistin-tolerant cells employ quorum sensing (QS) to initiate the formation of new colistin-tolerant subpopulations, highlighting multicellular behaviour in antibiotic tolerance development. The macrolide erythromycin, which has been previously shown to inhibit the motility and QS of P. aeruginosa, boosts biofilm eradication by colistin. Our work provides insights on the mechanisms underlying the formation of antibiotic-tolerant populations in bacterial biofilms and indicates research avenues for designing more efficient treatments against biofilm-associated infections.
Project description:Our current challenge in the management of prosthetic joint infection is the eradication of biofilms which has driven the need for improved antimicrobial agents and regimens. In this study, the antimicrobial peptide, LL-37, and silver nanoparticles (AgNPs) were investigated for their antimicrobial efficacies against Staphylococcus aureus (S. aureus), a microorganism commonly implicated in biofilm-related infections. These antimicrobials were compared to conventional antibiotics and combination treatments with rifampin. Using a Centers for Disease Control reactor, 24 h S. aureus biofilms were formed on cobalt-chromium discs and the anti-biofilm activity was determined by quantifying the amount of colony forming units following treatments. We found that LL-37 was the most efficacious antimicrobial agent with a more than 4 log reduction in colony counts. In comparison, silver nanoparticles and conventional antibiotics were not as efficacious, with a less than 1 log reduction in colony counts. Antimicrobial combination treatments with rifampin significantly increased the log reduction for AgNPs and gentamicin, although still significantly less than LL-37 in isolation. Furthermore, kinetic studies revealed the rapid elimination of S. aureus biofilm with LL-37. Collectively, the results of this study demonstrated that LL-37 was an effective agent against S. aureus biofilms and may have potential clinical applications in the eradication of biofilms and treatment of prosthetic joint infection.
Project description:Antimicrobial research is being pressured to look for more effective therapeutics for the ever-growing antibiotic-resistant infections, and antimicrobial peptides (AMP) and antimicrobial combinations are promising solutions. This work evaluates colistin-AMP combinations against two major pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, encompassing non- and resistant strains. Colistin (CST) combined with the AMP temporin A (TEMP-A), citropin 1.1 (CIT-1.1) and tachyplesin I linear analogue (TP-I-L) was tested against planktonic, single- and double-species biofilm cultures. Overall synergy for planktonic P. aeruginosa and synergy/additiveness for planktonic S. aureus were observed. Biofilm growth prevention was achieved with synergy and additiveness. Pre-established 24 h-old biofilms were harder to eradicate, especially for S. aureus and double-species biofilms; still, some synergy and addictiveness was observed for higher concentrations, including for the biofilms of resistant strains. Different treatment times and growth media did not greatly influence AMP activity. CST revealed low toxicity compared with the other AMP but its combinations were toxic for high concentrations. Overall, combinations reduced effective AMP concentrations, mainly in prevention scenarios. Improvement of effectiveness and toxicity of therapeutic strategies will be further investigated.