Identification of novel targets of azithromycin activity against Pseudomonas aeruginosa grown in physiologically relevant media.

Pseudomonas aeruginosa causes severe multidrug-resistant infections that often lead to bacteremia and sepsis. Physiologically relevant conditions can increase the susceptibility of pathogens to antibiotics, such as azithromycin (AZM). When compared to minimal-inhibitory concentrations (MICs) in laboratory media, AZM had a 16-fold lower MIC in tissue culture medium with 5% Mueller Hinton broth (MHB) and a 64-fold lower MIC in this tissue culture medium with 20% human serum. AZM also demonstrated increased synergy in combination with synthetic host-defense peptides DJK-5 and IDR-1018 under host-like conditions and in a murine abscess model. To mechanistically study the altered effects of AZM under physiologically relevant conditions, global transcriptional analysis was performed on P. aeruginosa with and without effective concentrations of AZM. This revealed that the arn operon, mediating arabinosaminylation of lipopolysaccharides and related regulatory systems, was down-regulated in host-like media when compared to MHB. Inactivation of genes within the arn operon led to increased susceptibility of P. aeruginosa to AZM and great increases in synergy between AZM and other antimicrobial agents, indicating that dysregulation of the arn operon might explain increased AZM uptake and synergy in host-like media. Furthermore, genes involved in central and energy metabolism and ribosome biogenesis were dysregulated more in physiologically relevant conditions treated with AZM, likely due to general changes in cell physiology as a result of the increased effectiveness of AZM in these conditions. These data suggest that, in addition to the arn operon, there are multiple factors in host-like environments that are responsible for observed changes in susceptibility.

Outer Membrane Interaction Kinetics of New Polymyxin B Analogs in Gram-Negative Bacilli.

Infections caused by drug-resistant Gram-negative bacilli are a severe global health threat, limiting effective drug choices for treatment. In this study, polymyxin analogs designed to have reduced nephrotoxicity, direct activity, and potentiating activity were assessed for inhibition and outer membrane interaction kinetics against wild-type (WT) and polymyxin or multidrug-resistant (MDR) Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae In MIC assays, two polymyxin B (PMB) analogs (SPR1205 and SPR206) and a polymyxin E analog (SPR946), with shortened peptide side chains and branched aminobutyryl N termini, exhibited promising activity compared with PMB and previously tested control polymyxin analogs SPR741 and polymyxin B nonapeptide (PMBN). Using dansyl-polymyxin (DPX) binding to assess the affinity of interaction with lipopolysaccharide (LPS), purified or in the context of intact cells, SPR206 exhibited similar affinities to PMB but higher affinities than the other SPR analogs. Outer membrane permeabilization measured by the 1-N-phenyl-napthylamine (NPN) assay did not differ significantly between the polymyxin analogs. Moreover, Hill numbers were greater than 1 for most of the compounds tested on E. coli and P. aeruginosa strains which indicates that the disruption of the outer membrane by one molecule of compound cooperatively enhances the subsequent interactions of other molecules against WT and MDR strains. The high activity demonstrated by SPR206 as well as its ability to displace LPS and permeabilize the outer membrane of multiple strains of Gram-negative bacilli while showing cooperative potential with other membrane disrupting compounds supports further research with this polymyxin analog.

Quick versus Quantitative: Evaluation of Two Commercial Real-Time PCR Assays for the Detection of Pneumocystis jirovecii from Bronchoalveolar Lavage Fluids.

Two commercial real-time PCR assays for the detection of Pneumocystis jirovecii were compared, the quantitative RealStar P. jirovecii assay and the qualitative DiaSorin P. jirovecii assay, the latter of which can be used without nucleic acid extraction. Archived bronchoalveolar lavage (BAL) specimens (n = 66), previously tested by molecular methods, were tested by both assays, and the results were compared to the respective original result. The RealStar P. jirovecii assay demonstrated good positive percent agreement (PPA) (90% [95% confidence interval (CI), 72 to 97%]; 27/30) and negative percent agreement (NPA) (100% [95% CI, 88 to 100%]; 36/36) with the reference method. The DiaSorin P. jirovecii assay concordantly detected P. jirovecii in 19 of 24 positive BAL samples (PPA = 73% [95% CI, 52 to 88%]). All negative BAL samples gave concordant results (NPA = 100% [95% CI, 87 to 100%]; 34/34). Discordant results occurred mostly in samples with low fungal loads. In conclusion, the RealStar assay demonstrated good concordance with reference results, and the DiaSorin P. jirovecii assay performed well for negative BAL and positive BAL samples with P. jirovecii concentrations of greater than 260 copies/mL. IMPORTANCE Pneumonia, caused by the opportunistic fungus Pneumocystis jirovecii, poses a significant risk for immunocompromised individuals. Laboratory testing for P. jirovecii is progressively shifting toward the use of molecular tests such as real-time PCR; however, this is often performed at reference laboratories. Many frontline laboratories are looking into improving their service and reducing turnaround times for obtaining P. jirovecii results by bringing molecular P. jirovecii testing in-house. We evaluated and compared two commercial real-time PCR assays with different workflows for the detection of P. jirovecii from bronchoalveolar lavage specimens. The RealStar P. jirovecii assay requires nucleic acid extraction and provides a quantification of fungal load for positive samples. The DiaSorin P. jirovecii assay offers a simple workflow without nucleic extraction from patient samples and qualitative results. Results from this study provide valuable information on performance and workflow considerations for laboratories that wish to implement P. jirovecii molecular testing.

Automated 16S Sequencing Using an R-Based Analysis Module for Bacterial Identification.

Sanger sequencing of the 16S rRNA gene is routinely used for the identification of bacterial isolates. However, this method is still performed mostly in more-specialized reference laboratories, and traditional protocols can be labor intensive. In this study, 99 clinical bacterial isolates were used to validate a fast, simplified, and largely automated protocol for 16S sequencing. The workflow combines real-time PCR of the first 500 bp of the bacterial 16S rRNA gene and amplicon sequencing on an automated, cartridge-based sequence analyzer. Sequence analysis, NCBI BLAST search, and result interpretation were performed using an automated R-based script. The automated workflow and R analysis described here produced results equal to those of manual sequence analysis. Of the 96 sequences with adequate quality, 90 were concordantly identified to the genus (n = 62) or species level (n = 28) compared with routine laboratory identification of the organism. One organism identification was discordant, and 5 resulted in an inconclusive identification. For sequences that gave a valid result, the overall accuracy of identification to at least the genus level was 98.9%. This simplified sequencing protocol provides a standardized approach to clinical 16S sequencing, analysis, and quality control that would be suited to frontline clinical microbiology laboratories with minimal experience. IMPORTANCE Sanger sequencing of the 16S rRNA gene is widely used as a diagnostic tool for bacterial identification, especially in cases where routine diagnostic methods fail to provide an identification, for organisms that are difficult to culture, or from specimens where cultures remain negative. Our simplified protocol is tailored toward use in frontline laboratories with little to no experience with sequencing. It provides a highly automated workflow that can deliver fast results with little hands-on time. Implementing 16S sequencing in-house saves additional time that is otherwise required to send out isolates/specimens for identification to reference laboratories. This makes results available much faster to physicians who can in turn initiate or adjust patient treatment accordingly.

Surviving the host: Microbial metabolic genes required for growth of Pseudomonas aeruginosa in physiologically-relevant conditions.

Pseudomonas aeruginosa, like other pathogens, adapts to the limiting nutritional environment of the host by altering patterns of gene expression and utilizing alternative pathways required for survival. Understanding the genes essential for survival in the host gives insight into pathways that this organism requires during infection and has the potential to identify better ways to treat infections. Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model. Genes known to be upregulated during infections, as well as those involved in nucleotide metabolism, and cobalamin (vitamin B12) biosynthesis, etc., were required for survival in vivo- and in host mimicking conditions, but not in nutrient rich lab medium, Mueller Hinton broth (MHB). Correspondingly, mutants in genes encoding proteins of nucleotide and cobalamin metabolism pathways were shown to have growth defects under physiologically-relevant media conditions, in vivo, and in vivo-like models, and were downregulated in expression under these conditions, when compared to MHB. This study provides evidence for the relevance of studying P. aeruginosa fitness in physiologically-relevant host mimicking conditions and identified metabolic pathways that represent potential novel targets for alternative therapies.

BosR: A novel biofilm-specific regulator in Pseudomonas aeruginosa.

Biofilms are the most common cause of bacterial infections in humans and notoriously hard to treat due to their ability to withstand antibiotics and host immune defenses. To overcome the current lack of effective antibiofilm therapies and guide future design, the identification of novel biofilm-specific gene targets is crucial. In this regard, transcriptional regulators have been proposed as promising targets for antimicrobial drug design. Therefore, a Transposon insertion sequencing approach was employed to systematically identify regulators phenotypically affecting biofilm growth in Pseudomonas aeruginosa PA14 using the TnSeq analysis tools Bio-TraDIS and TRANSIT. A screen of a pool of 300,000 transposon insertion mutants identified 349 genes involved in biofilm growth on hydroxyapatite, including 47 regulators. Detection of 19 regulatory genes participating in well-established biofilm pathways validated the results. An additional 28 novel prospective biofilm regulators suggested the requirement for multiple one-component transcriptional regulators. Biofilm-defective phenotypes were confirmed for five one-component transcriptional regulators and a protein kinase, which did not affect motility phenotypes. The one-component transcriptional regulator bosR displayed a conserved role in P. aeruginosa biofilm growth since its ortholog in P. aeruginosa strain PAO1 was also required for biofilm growth. Microscopic analysis of a chromosomal deletion mutant of bosR confirmed the role of this regulator in biofilm growth. Overall, our results highlighted that the gene network driving biofilm growth is complex and involves regulators beyond the primarily studied groups of two-component systems and cyclic diguanylate signaling proteins. Furthermore, biofilm-specific regulators, such as bosR, might constitute prospective new drug targets to overcome biofilm infections.

Testing physiologically relevant conditions in minimal inhibitory concentration assays.

The minimal inhibitory concentration (MIC) assay uses agar or broth dilution methods to measure, under defined test conditions, the lowest effective concentration of an antimicrobial agent that inhibits visible growth of a bacterium of interest. This assay is used to test the susceptibilities of bacterial isolates and of novel antimicrobial drugs, and is typically done in nutrient-rich laboratory media that have little relevance to in vivo conditions. As an extension to our original protocol on MIC assays (also published in Nature Protocols), here we describe the application of the MIC broth microdilution assay to test antimicrobial susceptibility in conditions that are more physiologically relevant to infections observed in the clinic. Specifically, we describe a platform that can be applied to the preparation of medium that mimics lung and wound exudate or blood conditions for the growth and susceptibility testing of bacteria, including ESKAPE pathogens. This protocol can also be applied to most physiologically relevant liquid medium and aerobic pathogens, and takes 3-4 d to complete.

Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application.

Highly antibiotic resistant, microbial communities, referred to as biofilms, cause various life-threatening infections in humans. At least two-thirds of all clinical infections are biofilm associated, and antibiotic therapy regularly fails to cure patients. Anti-biofilm peptides represent a promising approach to treat these infections by targeting biofilm-specific characteristics such as highly conserved regulatory mechanisms. They are being considered for clinical application and we discuss here key factors in discovery, design, and application, particularly the implementation of host-mimicking conditions, that are required to enable the successful advancement of potent anti-biofilm peptides from the bench to the clinic.

Alternative strategies for the study and treatment of clinical bacterial biofilms.

Biofilms represent an adaptive lifestyle where microbes grow as structured aggregates in many different environments, e.g. on body surfaces and medical devices. They are a profound threat in medical (and industrial) settings and cause two-thirds of all infections. Biofilm bacteria are especially recalcitrant to common antibiotic treatments, demonstrating adaptive multidrug resistance. For this reason, novel methods to eradicate or prevent biofilm infections are greatly needed. Recent advances have been made in exploring alternative strategies that affect biofilm lifestyle, inhibit biofilm formation, degrade biofilm components and/or cause dispersal. As such, naturally derived compounds, molecules that interfere with bacterial signaling systems, anti-biofilm peptides and phages show great promise. Their implementation as either stand-alone drugs or complementary therapies has the potential to eradicate resilient biofilm infections. Additionally, altering the surface properties of indwelling medical devices through bioengineering approaches has been examined as a method for preventing biofilm formation. There is also a need for improving current biofilm detection methods since in vitro methods often do not accurately measure live bacteria in biofilms or mimic in vivo conditions. We propose that the design and development of novel compounds will be enabled by the improvement and use of appropriate in vitro and in vivo models.