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.

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.

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.

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.

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.

The Inoculation Method Could Impact the Outcome of Microbiological Experiments.

For the past 150 years, bacteria have been investigated primarily in liquid batch cultures. Contrary to most expectations, these cultures are not homogeneous mixtures of single-cell bacteria, because free-floating bacterial aggregates eventually develop in most liquid batch cultures. These aggregates share characteristics with biofilms, such as increased antibiotic tolerance. We investigated how aggregates develop and what influences this development in liquid batch cultures of Pseudomonas aeruginosa We focused on how the method of inoculation affected aggregation by assessing aggregate frequency and size using confocal laser scanning microscopy. Several traditional methods of initiating an overnight bacterial culture, i.e., inoculation directly from frozen cultures, inoculation using agar-grown cells, or inoculation using cells grown in liquid cultures, were investigated. We discovered a direct link between the inoculation method and the size and frequency of biofilm aggregates in liquid batch cultures, with inoculation directly from a plate resulting in the most numerous and largest aggregates. These large aggregates had an overall impact on the cultures' subsequent tolerance toward tobramycin, indicating that the inoculation method has a profound impact on antibiotic tolerance. We also observed a mechanism whereby preformed aggregates recruited single cells from the surrounding culture in a "snowball effect," building up aggregated biomass in the culture. This recruitment was found to rely heavily on the exopolysaccharide Psl. Additionally, we found that both Escherichia coli and Staphylococcus aureus produced aggregates in liquid batch cultures. Our results stress the importance of inoculation consistency throughout experiments and the substantial impact aggregate development in liquid batch cultures may have on the outcomes of microbiological experiments.IMPORTANCE Pure liquid cultures are fundamental to the field of microbiological research. These cultures are normally thought of as homogeneous mixtures of single-cell bacteria; the present study shows that this is not always true. Bacteria may aggregate in these liquid cultures. The aggregation can be induced by the method chosen for inoculation. The presence of aggregates can significantly change the outcomes of experiments by altering the phenotype of the cultures. The study found a mechanism whereby preformed aggregates are able to recruit surrounding single cells in a form of snowball effect, creating more and larger aggregates in the cultures. Once formed, these aggregates are hard to remove. Aggregates in liquid cultures may be an immense unseen challenge for microbiologists.

Real-Time Monitoring of nfxB Mutant Occurrence and Dynamics in Pseudomonas aeruginosa Biofilm Exposed to Subinhibitory Concentrations of Ciprofloxacin.

Biofilm infections caused by Pseudomonas aeruginosa are frequently treated with ciprofloxacin (CIP); however, resistance rapidly develops. One of the primary resistance mechanisms is the overexpression of the MexCD-OprJ pump due to a mutation in nfxB, encoding the transcriptional repressor of this pump. The aim of this study was to investigate the effect of subinhibitory concentrations of CIP on the occurrence of nfxB mutants in the wild-type PAO1 flow cell biofilm model. For this purpose, we constructed fluorescent reporter strains (PAO1 background) with an mCherry tag for constitutive red fluorescence and chromosomal transcriptional fusion between the P mexCD promoter and gfp leading to green fluorescence upon mutation of nfxB We observed a rapid development of nfxB mutants by live confocal laser scanning microscopy (CLSM) imaging of the flow cell biofilm (reaching 80 to 90% of the whole population) when treated with 1/10 minimal biofilm inhibitory concentration of CIP for 24 h and 96 h. Based on the observed developmental stages, we propose that nfxB mutants emerged de novo in the biofilm during CIP treatment from filamentous cells, which might have arisen due to the stress responses induced by CIP. Identical nfxB mutations were found in fluorescent colonies from the same flow cell biofilm, especially in 24-h biofilms, suggesting selection and clonal expansion of the mutants during biofilm growth. Our findings point at the significant role of high-enough antibiotic dosages or appropriate combination therapy to avoid the emergence of resistant mutants in biofilms.

Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics.

Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 µm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3- as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3- as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment.IMPORTANCEPseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

Denitrification by cystic fibrosis pathogens - Stenotrophomonas maltophilia is dormant in sputum.

OBJECTIVE: Chronic Pseudomonas aeruginosa lung infection is the most severe complication for cystic fibrosis (CF) patients. Infected endobronchial mucus of CF patients contains anaerobic zones mainly due to the respiratory burst of polymorphonuclear leukocytes. We have recently demonstrated ongoing denitrification in sputum from patients infected with P. aeruginosa. Therefore we aimed to investigate, whether the pathogenicity of several known CF pathogens is correlated to their ability to perform denitrification. METHODS: We measured denitrification with N(2)O microsensors in concert with anaerobic growth measurements by absorbance changes and colony counting in isolates from 32 CF patients chronically infected with the highly pathogenic bacteria P. aeruginosa, Achromobacter xylosoxidans, Burkholderia multivorans or the less pathogenic bacterium Stenotrophomonas maltophilia. Consumption of NO(3)(-) and NO(2)(-) was estimated by the Griess Assay. All isolates were assayed during 2 days of incubation in anaerobic LB broth with NO(3)(-) or NO(2)(-). PNA FISH staining of 16S rRNA was used to estimate the amount of ribosomes per bacterial cells and thereby the in situ growth rate of S. maltophilia in sputum. RESULTS: Supplemental NO(3)(-) caused increased production of N(2)O by P. aeruginosa, A. xylosoxidans and B. multivorans and increased growth for all pathogens. Growth was, however, lowest for S. maltophilia. NO(3)(-) was metabolized by all pathogens, but only P. aeruginosa was able to remove NO(2)(-). S. maltophilia had limited growth in sputum as seen by the weak PNA FISH staining. CONCLUSIONS: All four pathogens were able to grow anaerobically by NO(3)(-) reduction. Denitrification as demonstrated by N(2)O production was, however, not found in S. maltophilia isolates. The ability to perform denitrification may contribute to the pathogenicity of the infectious isolates since complete denitrification promotes faster anaerobic growth. The inability of S. maltophilia to proliferate by denitrification and therefore grow in the anaerobic CF sputum may explain its low pathogenicity in CF patients.

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.

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.

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.

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.

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.

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.

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.

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).

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.