Adaptive Interactions of Achromobacter spp. with Pseudomonas aeruginosa in Cystic Fibrosis Chronic Lung Co-Infection.

In the lungs of patients with cystic fibrosis (CF), the main pathogen Pseudomonas aeruginosa is often co-isolated with other microbes, likely engaging in inter-species interactions. In the case of chronic co-infections, this cohabitation can last for a long time and evolve over time, potentially contributing to the clinical outcome. Interactions involving the emerging pathogens Achromobacter spp. have only rarely been studied, reporting inhibition of P. aeruginosa biofilm formation. To evaluate the possible evolution of such interplay, we assessed the ability of Achromobacter spp. isolates to affect the biofilm formation of co-isolated P. aeruginosa strains during long-term chronic co-infections. We observed both competition and cohabitation. An Achromobacter sp. isolate secreted exoproducts interfering with the adhesion ability of a co-isolated P. aeruginosa strain and affected its biofilm formation. Conversely, a clonal Achromobacter sp. strain later isolated from the same patient, as well as two longitudinal strains from another patient, did not show similar competitive behavior against its P. aeruginosa co-isolates. Genetic variants supporting the higher virulence of the competitive Achromobacter sp. isolate were found in its genome. Our results confirm that both inter-species competition and cohabitation are represented during chronic co-infections in CF airways, and evolution of these interplays can happen even at the late stages of chronic infection.

Enhanced Eradication of Mucin-Embedded Bacterial Biofilm by Locally Delivered Antibiotics in Functionalized Microcontainers.

Bacterial biofilm-related infections are difficult to eradicate and require repeated treatments with high doses of antibiotics. Thus, there is an urgent need for new treatment strategies that minimize the use of antibiotics while enhancing biofilm eradication. Functionalized reservoir-based microdevices, such as, microcontainers (MCs), offer, high drug loading capacity, mucus embedment, and tuneable drug release. Here, MCs are loaded with the antibiotic ciprofloxacin (CIP), and sealed with a lid consisting of chitosan (CHI) and a mucolytic agent, N-acetylcysteine (NAC). It is found that CHI and NAC work synergistically, showing improved mucoadhesive and mucolytic properties. To better mimic the in vivo habitat of Pseudomonas aeruginosa (P. aeruginosa), the biofilm is grown in a mucin-containing medium on a newly developed centrifugal microfluidic system. The CHI/NAC coated MCs improve eradication of biofilm (88.22 ± 2.89%) compared to CHI-coated MCs (72.68 ± 3.73%) or bolus injection (39.86 ± 13.28%). The findings suggest that MCs are significantly more efficient than a bolus treatment. Furthermore, CHI/NAC functionalized MCs kill most of the biomass already after 5 h (80.75 ± 3.50%), mainly due to a fast drug release. This is the first time that CHI/NAC has been combined as a coating to explore mucolytic properties on bacterial biofilms.

Bacterial persisters in long-term infection: Emergence and fitness in a complex host environment.

Despite intensive antibiotic treatment, Pseudomonas aeruginosa often persists in the airways of cystic fibrosis (CF) patients for decades, and can do so without antibiotic resistance development. Using high-throughput screening assays of bacterial survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of persisters in a large patient cohort using 460 longitudinal isolates of P. aeruginosa from 39 CF patients. Isolates were classed as high persister variants (Hip) if they regrew following antibiotic treatment in at least 75% of the experimental replicates. Strain genomic data, isolate phenotyping, and patient treatment records were integrated in a lineage-based analysis of persister formation and clinical impact. In total, 19% of the isolates were classified as Hip and Hip emergence increased over lineage colonization time within 22 Hip+ patients. Most Hip+ lineages produced multiple Hip isolates, but few Hip+ lineages were dominated by Hip. While we observed no strong signal of adaptive genetic convergence within Hip isolates, they generally emerged in parallel or following the development of ciprofloxacin resistance and slowed growth. Transient lineages were majority Hip-, while strains that persisted over a clinically diagnosed 'eradication' period were majority Hip+. Patients received indistinguishable treatment regimens before Hip emergence, but Hip+ patients overall were treated significantly more than Hip- patients, signaling repeated treatment failure. When subjected to in vivo-similar antibiotic dosing, a Hip isolate survived better than a non-Hip in a structured biofilm environment. In sum, the Hip phenotype appears to substantially contribute to long-term establishment of a lineage in the CF lung environment. Our results argue against the existence of a single dominant molecular mechanism underlying bacterial antibiotic persistence. We instead show that many routes, both phenotypic and genetic, are available for persister formation and consequent increases in strain fitness and treatment failure in CF airways.

Microcontainer Delivery of Antibiotic Improves Treatment of Pseudomonas aeruginosa Biofilms.

Biofilm-associated infections are difficult to treat effectively with antibiotics despite repeated treatments. Polymeric microdevices (microcontainers) have previously been shown to engulf in mucus layers and to provide tunable release. Such devices may overcome the challenge of delivering antibiotics into the biofilm, increasing the local drug concentration and hence improve local bacterial killing. In this work, microcontainers are loaded with the antibiotic, ciprofloxacin hydrochloride, and functionalized with polymeric lids of polyethylene glycol (PEG), chitosan, or Eudragit S100. The PEG lid gives rise to a drug release comparable to uncoated microcontainers showing complete release after 8 h, whereas chitosan and Eudragit S100 lids result in continuous release during the course of 24 h. All antibiotic-containing microcontainers inhibit planktonic growth of Pseudomonas aeruginosa (PAO1) cells, but the degree of inhibition depends on the coating. Microcontainers with ciprofloxacin hydrochloride kill about three times more biofilm-associated PAO1 cells compared with a single standard bolus. Moreover, the use of microcontainers in biofilm result in bacterial killing equal to a constant flow of a three times higher concentration of solubilized antibiotics. These studies suggest that microcontainers can be useful for antibiotic delivery in treatment of biofilm-associated infections, resulting in more effective treatment and reduced use of antibiotics.

Mutations causing low level antibiotic resistance ensure bacterial survival in antibiotic-treated hosts.

In 474 genome sequenced Pseudomonas aeruginosa isolates from 34 cystic fibrosis (CF) patients, 40% of these harbor mutations in the mexZ gene encoding a negative regulator of the MexXY-OprM efflux pump associated with aminoglycoside and fluoroquinolone resistance. Surprisingly, resistance to aminoglycosides and fluoroquinolones of mexZ mutants was far below the breakpoint of clinical resistance. However, the fitness increase of the mutant bacteria in presence of the relevant antibiotics, as demonstrated in competition experiments between mutant and ancestor bacteria, showed that 1) very small phenotypic changes cause significant fitness increase with severe adaptive consequences, and 2) standardized phenotypic tests fail to detect such low-level variations. The frequent appearance of P. aeruginosa mexZ mutants in CF patients is directly connected to the intense use of the target antibiotics, and low-level antibiotic resistance, if left unnoticed, can result in accumulation of additional genetic changes leading to high-level resistance.

Determination of Tobramycin in M9 Medium by LC-MS/MS: Signal Enhancement by Trichloroacetic Acid.

It is well known that ion-pairing reagents cause ion suppression in LC-MS/MS methods. Here, we report that trichloroacetic acid increases the MS signal of tobramycin. To support studies of an in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms, an LC-MS/MS method for determination of tobramycin in M9 media was developed. Aliquots of 25 µL M9 media samples were mixed with the internal standard (IS) tobramycin-d5 (5 µg/mL, 25 µL) and 200 µL 2.5% trichloroacetic acid. The mixture (5 µL) was directly injected onto a PFP column (2.0 × 50 mm, 3 µm) eluted with water containing 20 mM ammonium formate and 0.14% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid in a gradient mode. ESI+ and MRM with ion m/z 468 → 324 for tobramycin and m/z 473 → 327 for the IS were used for quantification. The calibration curve concentration range was 50-25000 ng/mL. Matrix effect from M9 media was not significant when compared with injection solvents, but signal enhancement by trichloroacetic acid was significant (∼3 fold). The method is simple, fast, and reliable. Using the method, the in vitro PK/PD model was tested with one bolus dose of tobramycin.

Physical and sensory characteristics of pork sausages from enzymatically modified blends of lard and rapeseed oil during storage.

Physical and sensory characteristic of pork sausages produced from enzymatic interesterified blends of lard and rapeseed oil during storage were evaluated. All three enzymatic interesterified blends (IE90, IE70 and IE50) had ratios of unsaturated to saturated fatty acids within the range of 1.47-2.84 which is favourable for cardiovascular disease risk reduction. Blends of IE90 and IE70 were found to have suitable solid fat content, melting and crystallization profile suitable for sausages production. Sausages were produced from blends of IE90 and IE70 with different muscle types (musculus longissimus dorsi and musculus sternomandibularis) and processing conditions such as cooling rates and final processing temperature. Cooling rate was found to have no significant (P>0.05) effect on hardness of the sausages throughout storage. Both musculus longissimus dorsi and high final processing temperature of 20 degrees C increased the hardness of the sausages during storage. In terms of fat particle size distribution, it was found that sausages IE70 had significantly (P<0.05) lower amount of small fat particles (<4 microm) and higher amount of big fat particles (4-500 microm). This is in agreement with the findings on softer texture of sausages IE70. All the sausages produced from interesterified blends of lard and rapeseed oil had no apparent fat excretion and were rated as having acceptable sensory attributes as compared to reference sausage which was produced from pure lard.

Culture conditions of Roseobacter strain 27-4 affect its attachment and biofilm formation as quantified by real-time PCR.

The fish probiotic bacterium Roseobacter strain 27-4 grows only as rosettes and produces its antibacterial compound under static growth conditions. It forms three-dimensional biofilms when precultured under static conditions. We quantified attachment of Roseobacter strain 27-4 using a direct real-time PCR method and demonstrated that the bacteria attached more efficiently to surfaces during static growth than under aerated conditions.