The role of individual exopolysaccharides in antibiotic tolerance of Pseudomonas aeruginosa aggregates.

The bacterium Pseudomonas aeruginosa is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria. Here, we investigated the role of individual P. aeruginosa exopolysaccharides in aggregate-associated antibiotic tolerance. We employed an aggregate-based antibiotic tolerance assay on a set of P. aeruginosa strains that were genetically engineered to over-produce a single, none, or all of the three exopolysaccharides Pel, Psl, and alginate. The antibiotic tolerance assays were conducted with the clinically relevant antibiotics tobramycin, ciprofloxacin and meropenem. Our study suggests that alginate plays a role in the tolerance of P. aeruginosa aggregates toward tobramycin and meropenem, but not ciprofloxacin. However, contrary to previous studies we did not observe a role for Psl or Pel in the tolerance of P. aeruginosa aggregates toward tobramycin, ciprofloxacin, and meropenem.

Protocol to assess metabolic activity of Pseudomonas aeruginosa by measuring heat flow using isothermal calorimetry.

Here, we present a protocol for assessing metabolic activity of bacterial populations by measuring heat flow using isothermal calorimetry. We outline the steps for preparing the different growth models of Pseudomonas aeruginosa and performing continuous metabolic activity measurements in the calScreener. We detail simple principal component analysis to differentiate between metabolic states of different populations and probabilistic logistic classification to assess resemblance to wild-type bacteria. This protocol for fine-scale metabolic measurement can aid in understanding microbial physiology. For complete details on the use and execution of this protocol, please refer to Lichtenberg et al. (2022).1.

Colistin Enhances Rifampicin's Antimicrobial Action in Colistin-Resistant Pseudomonas aeruginosa Biofilms.

The emergence of multidrug-resistant Pseudomonas aeruginosa infections has urged the need to find new strategies, such as the use of combinations of antibiotics. Among these, the combination of colistin with other antibiotics has been studied. Here, the action of combinations of colistin and rifampicin on both planktonic and sessile cells of colistin-resistant P. aeruginosa was studied. Dynamic biofilms were formed and treated with such a combination, resulting in an active killing effect of both colistin-resistant and colistin-susceptible P. aeruginosa in biofilms. The results suggest that the action of colistin on the outer membrane facilitates rifampicin penetration, regardless of the colistin-resistant phenotype. Based on these in vitro data, we propose a colistin-rifampicin combination as a promising treatment for infections caused by colistin-resistant P. aeruginosa.

3D blood-brain barrier-organoids as a model for Lyme neuroborreliosis highlighting genospecies dependent organotropism.

Lyme neuroborreliosis (LNB), a tick-borne infection caused by spirochetes within the Borrelia burgdorferi sensu lato (s.L.) complex, is among the most prevalent bacterial central nervous system (CNS) infections in Europe and the US. Here we have screened a panel of low-passage B. burgdorferi s.l. isolates using a novel, human-derived 3D blood-brain barrier (BBB)-organoid model. We show that human-derived BBB-organoids support the entry of Borrelia spirochetes, leading to swelling of the organoids and a loss of their structural integrity. The use of the BBB-organoid model highlights the organotropism between B. burgdorferi s.l. genospecies and their ability to cross the BBB contributing to CNS infection.

Synergy between Mecillinam and Ceftazidime/Avibactam or Avibactam against Multi-Drug-Resistant Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae.

Background: Carbapenemase-producing Klebsiella pneumoniae and Escherichia coli have become a significant global health challenge. This has created an urgent need for new treatment modalities. We evaluated the efficacy of mecillinam in combination with either avibactam or ceftazidime/avibactam against carbapenemase-producing clinical isolates. Materials and methods: Nineteen MDR clinical isolates of K. pneumoniae and E. coli were selected for the presence of blaKPC, blaNDM, blaOXA or blaIMP based on whole-genome sequencing and phenotypic susceptibility testing. We tested the synergy between mecillinam and avibactam or ceftazidime/avibactam. We used time−kill studies in vitro and a mouse peritonitis/sepsis model to confirm the synergistic effect. We investigated avibactam's impact on mecillinam´s affinity for penicillin-binding proteins with a Bocillin assay, and cell changes with phase-contrast and confocal laser scanning microscopy. Results: Mecillinam combined with ceftazidime/avibactam or avibactam substantially reduced MICs (from up to >256 µg/mL to <0.0016 µg/mL) for 17/18 strains. Significant log-CFU reductions were confirmed in time−kill and in vivo experiments. The Bocillin assay did not reveal changes. Conclusion: Mecillinam in combination with avibactam or ceftazidime/avibactam has a notable effect on most types of CPEs, both in vitro and in vivo. The mecillinam/avibactam combination treatment could be a new efficient antibiotic treatment against multi-drug-resistant carbapenemase-producing Gram-negative pathogens.

Cyclic-di-GMP signaling controls metabolic activity in Pseudomonas aeruginosa.

Bacteria in biofilms are embedded in extracellular matrix and display low metabolic activity, partly due to insufficient diffusive exchange of metabolic substrate. The extracellular matrix and low metabolic activity both contribute to the high antibiotic tolerance-the hallmark of biofilm bacteria. The second messenger molecule, c-di-GMP, regulates biofilm development in Pseudomonas aeruginosa, where high internal levels lead to biofilm formation and low levels are associated with planktonic bacteria. Using a microcalorimetric approach, we show that c-di-GMP signaling is a major determinant of the metabolic activity of P. aeruginosa, both in planktonic culture and in two biofilm models. The high c-di-GMP content of biofilm bacteria forces them to rapidly spend a large amount of energy on the production of exopolysaccharides, resulting in a subsequent low metabolic state. This suggests that the low metabolic state of bacteria in mature biofilms, to some extent, is a consequence of a c-di-GMP-regulated survival strategy.

Isothermal microcalorimetry vs checkerboard assay to evaluate in-vitro synergism of meropenem-amikacin and meropenem-colistin combinations against multi-drug-resistant Gram-negative pathogens.

OBJECTIVES: To evaluate the activity of meropenem-amikacin and meropenem-colistin combinations with checkerboard broth microdilution (CKBM) compared with isothermal microcalorimetry (ITMC) assays against a multi-centric collection of multi-drug-resistant Gram-negative clinical isolates; and to compare the fractional inhibitory concentration (FIC) index and time to results of CKBM and ITMC. METHODS: A collection of 333 multi-drug-resistant Gram-negative clinical isolates showing reduced susceptibility to meropenem (121 Klebsiella pneumoniae, 14 Escherichia coli, 130 Pseudomonas aeruginosa and 68 Acinetobacter baumannii) isolated from different centres (Florence, Madrid, Rotterdam and Stockholm) was included in the study. The antimicrobial activity of meropenem-amikacin and meropenem-colistin combinations was evaluated with CKBM and ITMC. FIC index results were interpreted as synergistic/additive and indifferent for values ≤0.5/0.51, respectively. Whole-genome sequencing data of a subset of strains were used to evaluate their clonality. RESULTS: In total, 254 and 286 strains were tested with meropenem-colistin and meropenem-amikacin combinations with ITMC and CKBM, respectively. Synergistic/additive effects were observed for 46 strains (20 K. pneumoniae, four E. coli, 22 P. aeruginosa) and 20 strains (three K. pneumoniae, 11 P. aeruginosa and six A. baumannii) with meropenem-amikacin and meropenem-colistin combinations, respectively, with CKBM. ITMC showed good concordance with CKBM, with 89.5% and 92.2% of cases interpreted within the same FIC index category for meropenem-amikacin and meropenem-colistin combinations, respectively. Most of the synergistic/additive effects were detected within 6 h by ITMC. CONCLUSIONS: ITMC showed very good concordance with CKBM against a large collection of multi-drug-resistant Gram-negative clinical isolates, and could be implemented for the rapid evaluation of in-vitro activity of antimicrobial combinations.

Transcription of the Alginate Operon in Pseudomonas aeruginosa Is Regulated by c-di-GMP.

Overproduction of the exopolysaccharide alginate contributes to the pathogenicity and antibiotic tolerance of Pseudomonas aeruginosa in chronic infections. The second messenger, c-di-GMP, is a positive regulator of the production of various biofilm matrix components and is known to regulate alginate synthesis at the posttranslational level in P. aeruginosa. We provide evidence that c-di-GMP also regulates transcription of the alginate operon in P. aeruginosa. Previous work has shown that transcription of the alginate operon is regulated by nine different proteins, AmrZ, AlgP, IHFα, IHFß, CysB, Vfr, AlgR, AlgB, and AlgQ, and we investigated if some of these proteins function as a c-di-GMP effector. We found that deletion of algP, algQ, IHFα, and IHFß had only a marginal effect on the transcription of the alginate operon. Deletion of vfr and cysB led to decreased transcription of the alginate operon, and the dependence of the c-di-GMP level was less pronounced, indicating that Vfr and CysB could be partially required for c-di-GMP-mediated regulation of alginate operon transcription. Our experiments indicated that the AmrZ, AlgR, and AlgB proteins are absolutely required for transcription of the alginate operon. However, differential radial capillary action of ligand assay (DRaCALA) and site-directed mutagenesis indicated that c-di-GMP does not bind to any of the AmrZ, AlgR, and AlgB proteins. IMPORTANCE The proliferation of alginate-overproducing P. aeruginosa variants in the lungs of cystic fibrosis patients often leads to chronic infection. The alginate functions as a biofilm matrix that protects the bacteria against host immune defenses and antibiotic treatment. Knowledge about the regulation of alginate synthesis is important in order to identify drug targets for the development of medicine against chronic P. aeruginosa infections. We provide evidence that c-di-GMP positively regulates transcription of the alginate operon in P. aeruginosa. Moreover, we revisited the role of the known alginate regulators, AmrZ, AlgP, IHFα, IHFß, CysB, Vfr, AlgR, AlgB, and AlgQ, and found that their effect on transcription of the alginate operon is highly varied. Deletion of algP, algQ, IHFα, or IHFß only had a marginal effect on transcription of the alginate operon, whereas deletion of vfr or cysB led to decreased transcription and deletion of amrZ, algR, or algB abrogated transcription.

A novel Borrelia-specific real-time PCR assay is not suitable for diagnosing Lyme neuroborreliosis.

BACKGROUND: Diagnosing Lyme neuroborreliosis (LNB) is complicated by a lack of adequate test systems and by the complex culturing conditions required to grow the causative pathogens in the Borrelia sensu lato complex. Improved testing methods are urgently needed. Here, we evaluate the applicability of a novel commercially available Borrelia-specific real-time PCR assay to diagnose LNB. MATERIALS AND METHODS: The specificity and sensitivity of the novel alphaCube Borrelia real-time PCR assay (Mikrogen) and the well-tested Micro-Dx™ real-time PCR assay (Molzym) were evaluated in cerebrospinal fluid (CSF) spiked with known amounts of Borrelia garinii and CSF from 19 patients with definite or possible LNB. CSF from patients diagnosed with neurosyphilis or enterovirus meningitis served as controls. RESULTS: The alphaCube assay specifically identified Borrelia down to 93 B garinii cells/mL in spiked CSF samples. The Micro-Dx™ real-time PCR assay was able to identify the presence of bacteria down to 9300 cells/mL in spiked samples. In CSF from patients diagnosed with LNB the sensitivity of the alphaCube assay was 0.00 and 0.00 for the Micro-DX. CONCLUSION: Although the alphaCube Borrelia assay was able to identify down to 93 cells/mL in spiked CSF samples, the inability to identify Borrelia in CSF samples from patients with LNB suggests that this type of infection carries a bacterial load in CSF below this detection level. Based on these results, neither the alphaCube Borrelia real-time PCR assay nor the Micro-Dx™ real-time PCR assay can be recommended for routine diagnostics of LNB using CSF samples.

Bacterial biofilms predominate in both acute and chronic human lung infections.

BACKGROUND: A basic paradigm of human infection is that acute bacterial disease is caused by fast growing planktonic bacteria while chronic infections are caused by slow-growing, aggregated bacteria, a phenomenon known as a biofilm. For lung infections, this paradigm has been thought to be supported by observations of how bacteria proliferate in well-established growth media in the laboratory-the gold standard of microbiology. OBJECTIVE: To investigate the bacterial architecture in sputum from patients with acute and chronic lung infections. METHODS: Advanced imaging technology was used for quantification and direct comparison of infection types on fresh sputum samples, thereby directly testing the acute versus chronic paradigm. RESULTS: In this study, we compared the bacterial lifestyle (planktonic or biofilm), growth rate and inflammatory response of bacteria in freshly collected sputum (n=43) from patient groups presenting with acute or chronic lung infections. We found that both acute and chronic lung infections are dominated by biofilms (aggregates of bacteria within an extracellular matrix), although planktonic cells were observed in both sample types. Bacteria grew faster in sputum from acute infections, but these fast-growing bacteria were enriched in biofilms similar to the architecture thought to be reserved for chronic infections. Cellular inflammation in the lungs was also similar across patient groups, but systemic inflammatory markers were only elevated in acute infections. CONCLUSIONS: Our findings indicate that the current paradigm of equating planktonic with acute and biofilm with chronic infection needs to be revisited as the difference lies primarily in metabolic rates, not bacterial architecture.

Biofilms can act as plasmid reserves in the absence of plasmid specific selection.

Plasmids facilitate rapid bacterial adaptation by shuttling a wide variety of beneficial traits across microbial communities. However, under non-selective conditions, maintaining a plasmid can be costly to the host cell. Nonetheless, plasmids are ubiquitous in nature where bacteria adopt their dominant mode of life - biofilms. Here, we demonstrate that biofilms can act as spatiotemporal reserves for plasmids, allowing them to persist even under non-selective conditions. However, under these conditions, spatial stratification of plasmid-carrying cells may promote the dispersal of cells without plasmids, and biofilms may thus act as plasmid sinks.

Peptidoglycan-Binding Anchor Is a Pseudomonas aeruginosa OmpA Family Lipoprotein With Importance for Outer Membrane Vesicles, Biofilms, and the Periplasmic Shape.

The outer membrane protein A (OmpA) family contains an evolutionary conserved domain that links the outer membrane in Gram-negative bacteria to the semi-rigid peptidoglycan (PG) layer. The clinically significant pathogen Pseudomonas aeruginosa carries several OmpA family proteins (OprF, OprL, PA0833, and PA1048) that share the PG-binding domain. These proteins are important for cell morphology, membrane stability, and biofilm and outer membrane vesicle (OMV) formation. In addition to other OmpAs, in silico analysis revealed that the putative outer membrane protein (OMP) with gene locus PA1041 is a lipoprotein with an OmpA domain and, hence, is a potential virulence factor. This study aimed to evaluate PA1041 as a PG-binding protein and describe its effect on the phenotype. Clinical strains were confirmed to contain the lipoprotein resulting from PA1041 expression with Western blot, and PG binding was verified in enzyme-linked immunosorbent assay (ELISA). By using a Sepharose bead-based ELISA, we found that the lipoprotein binds to meso-diaminopimelic acid (mDAP), an amino acid in the pentapeptide portion of PGs. The reference strain PAO1 and the corresponding transposon mutant PW2884 devoid of the lipoprotein were examined for phenotypic changes. Transmission electron microscopy revealed enlarged periplasm spaces near the cellular poles in the mutant. In addition, we observed an increased release of OMV, which could be confirmed by nanoparticle tracking analysis. Importantly, mutants without the lipoprotein produced a thick, but loose and unorganized, biofilm in flow cells. In conclusion, the lipoprotein from gene locus PA1041 tethers the outer membrane to the PG layer, and mutants are viable, but display severe phenotypic changes including disordered biofilm formation. Based upon the phenotype of the P. aeruginosa PW2884 mutant and the function of the protein, we designate the lipoprotein with locus tag PA1041 as "peptidoglycan-binding anchor" (Pba).

Induction of Native c-di-GMP Phosphodiesterases Leads to Dispersal of Pseudomonas aeruginosa Biofilms.

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation, whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. c-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases. We previously presented evidence that the ectopic expression of the Escherichia coli phosphodiesterase YhjH in Pseudomonas aeruginosa results in biofilm dispersal. More recently, however, evidence has been presented that the induction of native c-di-GMP phosphodiesterases does not lead to a dispersal of P. aeruginosa biofilms. The latter result may discourage attempts to use c-di-GMP signaling as a target for the development of antibiofilm drugs. However, here, we demonstrate that the induction of the P. aeruginosa c-di-GMP phosphodiesterases PA2133 and BifA indeed results in the dispersal of P. aeruginosa biofilms in both a microtiter tray biofilm assay and a flow cell biofilm system.

Effective antimicrobial combination in vivo treatment predicted with microcalorimetry screening.

OBJECTIVES: The worldwide emergence of antibiotic resistance calls for effective exploitation of existing antibiotics. Antibiotic combinations with different modes of action can synergize for successful treatment. In the present study, we used microcalorimetry screening to identify synergistic combination treatments against clinical MDR isolates. The synergistic effects were validated in a murine infection model. METHODS: The synergy of meropenem combined with colistin, rifampicin or amikacin was tested on 12 isolates (1 Escherichia coli, 5 Klebsiella pneumoniae, 3 Pseudomonas aeruginosa and 3 Acinetobacter baumannii) in an isothermal microcalorimeter measuring metabolic activity. One A. baumannii strain was tested with two individual pairings of antibiotic combinations. The microcalorimetric data were used to predict in vivo efficacy in a murine peritonitis/sepsis model. NMRI mice were inoculated intraperitoneally and after 1 h treated with saline, drug X, drug Y or X+Y. Bacterial load was determined by cfu in peritoneal fluid and blood after 4 h. RESULTS: In vitro, of the 13 combinations tested on the 12 strains, 3 of them exhibited a synergistic reduction in MIC (23% n = 3/13), 5 showed an additive effect (38.5% n = 5/13) and 5 had indifferent or antagonistic effects (38.5% n = 5/13). There was a significant correlation (P = 0.024) between microcalorimetry-screening FIC index values and the log reduction in peritoneal fluid from mice that underwent combination treatment compared with the most effective mono treatment. No such correlation could be found between chequerboard and in vivo results (P = 0.16). CONCLUSIONS: These data support microcalorimetic metabolic readout to predict additive or synergistic effects of combination treatment of MDR infections within hours.

The origin of extracellular DNA in bacterial biofilm infections in vivo.

Extracellular DNA (eDNA) plays an important role in both the aggregation of bacteria and in the interaction of the resulting biofilms with polymorphonuclear leukocytes (PMNs) during an inflammatory response. Here, transmission electron and confocal scanning laser microscopy were used to examine the interaction between biofilms of Pseudomonas aeruginosa and PMNs in a murine implant model and in lung tissue from chronically infected cystic fibrosis patients. PNA FISH, DNA staining, labeling of PMN DNA with a thymidine analogue and immunohistochemistry were applied to localize bacteria, eDNA, PMN-derived eDNA, PMN-derived histone H3 (H3), neutrophil elastase (NE) and citrullinated H3 (citH3). Host-derived eDNA was observed surrounding bacterial biofilms but not within the biofilms. H3 localized to the lining of biofilms while NE was found throughout biofilms. CitH3, a marker for neutrophil extracellular traps (NETs) was detected only sporadically indicating that most host-derived eDNA in vivo was not a result of NETosis. Together these observations show that, in these in vivo biofilm infections with P. aeruginosa, the majority of eDNA is found external to the biofilm and derives from the host.

In vivo demonstration of Pseudomonas aeruginosa biofilms as independent pharmacological microcompartments.

BACKGROUND: Pseudomonas aeruginosa is difficult to eradicate from the lungs of cystic fibrosis (CF) patients due to biofilm formation. Organs and blood are independent pharmacokinetic (PK) compartments. Previously, we showed in vitro biofilms behave as independent compartments impacting the pharmacodynamics. The present study investigated this phenomenon in vivo. METHODS: Seaweed alginate beads with P. aeruginosa resembling biofilms, either freshly produced (D0) or incubated for 5 days (D5) were installed s.c in BALB/c mice. Mice (n = 64) received tobramycin 40 mg/kg s.c. and were sacrificed at 0.5, 3, 6, 8, 16 or 24 h after treatment. Untreated controls (n = 14) were sacrificed, correspondingly. Tobramycin concentrations were determined in serum, muscle tissue, lung tissue and beads. Quantitative bacteriology was determined. RESULTS: The tobramycin peak concentrations in serum was 58.3 (±9.2) mg/L, in lungs 7.1 mg/L (±2.3), muscle tissue 2.8 mg/L (±0.5) all after 0.5 h and in D0 beads 19.8 mg/L (±3.5) and in D5 beads 24.8 mg/L (±4.1) (both 3 h). A 1-log killing of P. aeruginosa in beads was obtained at 8h, after which the bacterial level remained stable at 16 h and even increased in D0 beads at 24 h. Using the established diffusion retardation model the free tobramycin concentration inside the beads showed a delayed buildup of 3 h but remained lower than the MIC throughout the 24 h. CONCLUSIONS: The present in vivo study based on tobramycin exposure supports that biofilms behave as independent pharmacological microcompartments. The study indicates, reducing the biofilm matrix would increase free tobramycin concentrations and improve therapeutic effects.

Antibiotic susceptibility of cystic fibrosis lung microbiome members in a multispecies biofilm.

The lungs of cystic fibrosis (CF) patients are often chronically colonized by multiple microbial species that can form biofilms, including the major CF pathogen Pseudomonas aeruginosa. 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 P. aeruginosa influence microbial diversity. To this end, a microaerophilic multispecies biofilm model of frequently co-isolated members of the CF lung microbiome (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans) 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 P. aeruginosa. 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 (Prevotella melaninogenica, Veillonella parvula, and Fusobacterium nucleatum) 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.

Biofilms of Mycobacterium abscessus Complex Can Be Sensitized to Antibiotics by Disaggregation and Oxygenation.

Pulmonary infection with the multidrug-resistant Mycobacterium abscessus complex (MABSC) is difficult to treat in individuals with cystic fibrosis (CF). MABSC grows as biofilm aggregates in CF patient lungs, which are known to have anaerobic niches. How aggregation and anoxic conditions affect antibiotic tolerance is not well understood. We sought to determine whether disaggregation and oxygen availability sensitize MABSC isolates to recommended antibiotics. We tested the susceptibilities of 33 isolates from 22 CF patients with MABSC infection and a reference strain to the following antibiotics: amikacin, azithromycin, cefoxitin, ciprofloxacin, clarithromycin, imipenem, kanamycin, linezolid, moxifloxacin, rifampin, tigecycline, and sulfamethoxazole-trimethoprim. Isolates were grown in Mueller-Hinton broth with and without the disaggregating detergent Tween 80 (5%). Time-kill curves at days 1 and 3 were generated for oxic and anoxic amikacin treatment in 4-fold dilutions ranging from 2 to 512 mg liter-1 Scanning electron microscopy was used to visualize the aggregation patterns, while confocal laser scanning microscopy and microrespirometry were used to visualize biofilm growth patterns. Disruption of MABSC aggregates increased susceptibility to amikacin, tigecycline, kanamycin, azithromycin, imipenem, cefoxitin, and clarithromycin (P < 0.05, n = 29 to 31). Oxygenation enhanced the killing of disaggregated MABSC isolates by amikacin (P < 0.05) by 1 to 6 log units when 2 to 512 mg liter-1 of amikacin was used. This study explains why current drug susceptibility testing results correlate poorly with treatment outcomes. The conditions achieved by oxic culturing of planktonic isolates in vitro do not resemble the hypoxic conditions in CF patient lungs. Biofilm disruption and increased O2 availability during antibiotic therapy may be new therapeutic strategies for chronic MABSC infection.

In Situ Monitoring of the Antibacterial Activity of a Copper-Silver Alloy Using Confocal Laser Scanning Microscopy and pH Microsensors.

The antibacterial efficacy of a copper-silver alloy coating under conditions resembling build up of dry surface bacterial biofilms is successfully demonstrated according to US EPA test methods with a ≥99.9% reduction of test organisms over a 24 h period. A tailor-made confocal imaging protocol is designed to visualize in situ the killing of bacterial biofilms at the copper-silver alloy surface and monitor the kinetics for 100 min. The copper-silver alloy coating eradicates a biofilm of Gram-positive bacteria within 5 min while a biofilm of Gram-negative bacteria are killed more slowly. In situ pH monitoring indicates a 2-log units increase at the interface between the metallic surface and bacterial biofilm; however, the viability of the bacteria is not directly affected by this raise (pH 8.0-9.5) when tested in buffer. The OH- production, as a result of the interaction between the electrochemically active surface and the bacterial biofilm under environmental conditions, is thus one aspect of the contact-mediated killing of the copper-silver alloy coating and not the direct cause of the observed antibacterial efficacy. The combination of oxidation of bacterial cells, release of copper ions, and local pH raise characterizes the antibacterial activity of the copper-silver alloy-coated dry surface.

Is pseudarthrosis after spinal instrumentation caused by a chronic infection?

HYPOTHESIS: To assess whether a chronic bacterial infection is present in a subset of patients with pseudarthrosis after instrumented spinal fusion. METHODS: This was a prospective diagnostic study including adult patients with previous instrumented spinal fusion. Patients underwent revision surgery for either pseudarthrosis or other causes (e.g. implant removal, curve progression or junctional kyphosis) (control group). Five separate biopsies were randomly collected, intraoperatively, from the pseudarthrosis site and cultivated under both aerobic (5 days) and anaerobic (14 days) conditions. If cultivation was positive in at least 2/5 tissue samples, the biopsy was sectioned and stained using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). Confocal laser scanning microscopy was used to examine the sections and visualize bacterial aggregates. RESULTS: The study included 32 pseudarthrosis and 32 control patients. Cultivation yielded bacteria in at least 1/5 biopsies in 52% of patients with no difference between the groups (p = 1.0). Bacteria of the same species was found in at least 2/5 samples in seven pseudarthrosis patients and four controls (p = 0.509). Propionibacterium acnes was found in 8 of these 11 samples. Microscopy demonstrated tissue-embedded bacterial aggregates in two of these patients but with no inflammatory cells indicating an active infection. The presence of bacteria was not associated with the number of previous spinal procedures or the pre-revision fusion length (p ≥ 0.503). CONCLUSIONS: Pseudarthrosis after instrumented spinal surgery was not significantly associated with the presence of bacteria at the pseudarthrosis site. Positive cultivation results are common after spinal instrumentation, but our results indicate that they rarely represent an organized infection. These slides can be retrieved under Electronic Supplementary Material.