Synergistic effects of inhaled aztreonam plus tobramycin on hypermutable cystic fibrosis Pseudomonas aeruginosa isolates in a dynamic biofilm model evaluated by mechanism-based modelling and whole genome sequencing.

OBJECTIVE: Hypermutable Pseudomonas aeruginosa strains are highly prevalent in chronic lung infections of patients with cystic fibrosis (CF). Acute exacerbations of these infections have limited treatment options. This study aimed to investigate inhaled aztreonam and tobramycin against clinical hypermutable P. aeruginosa strains using the CDC dynamic in vitro biofilm reactor (CBR), mechanism-based mathematical modelling (MBM) and genomic studies. METHODS: Two CF multidrug-resistant strains were investigated in a 168 h CBR (n = 2 biological replicates). Regimens were inhaled aztreonam (75 mg 8-hourly) and tobramycin (300 mg 12-hourly) in monotherapies and combination. The simulated pharmacokinetic profiles of aztreonam and tobramycin (t1/2 = 3 h) were based on published lung fluid concentrations in patients with CF. Total viable and resistant counts were determined for planktonic and biofilm bacteria. MBM of total and resistant bacterial counts and whole genome sequencing were completed. RESULTS: Both isolates showed reproducible bacterial regrowth and resistance amplification for the monotherapies by 168 h. The combination performed synergistically, with minimal resistant subpopulations compared to the respective monotherapies at 168 h. Mechanistic synergy appropriately described the antibacterial effects of the combination regimen in the MBM. Genomic analysis of colonies recovered from monotherapy regimens indicated noncanonical resistance mechanisms were likely responsible for treatment failure. CONCLUSION: The combination of aztreonam and tobramycin was required to suppress the regrowth and resistance of planktonic and biofilm bacteria in all biological replicates of both hypermutable multidrug-resistant P. aeruginosa CF isolates. The developed MBM could be utilised for future investigations of this promising inhaled combination.

Pathogenomic analysis and characterization of Pasteurella multocida strains recovered from human infections.

Pasteurella multocida is an upper respiratory tract commensal in several mammal and bird species but can also cause severe disease in humans and in production animals such as poultry, cattle, and pigs. In this study, we performed whole-genome sequencing of P. multocida isolates recovered from a range of human infections, from the mouths of cats, and from wounds on dogs. Together with publicly available P. multocida genome sequences, we performed phylogenetic and comparative genomic analyses. While isolates from cats and dogs were spread across the phylogenetic tree, human infections were caused almost exclusively by subsp. septica strains. Most of the human isolates were capsule type A and LPS type L1 and L3; however, some strains lacked a capsule biosynthesis locus, and some strains contained a novel LPS outer-core locus, distinct from the eight LPS loci that can currently be identified using an LPS multiplex PCR. In addition, the P. multocida strains isolated from human infections contained novel mobile genetic elements. We compiled a curated database of known P. multocida virulence factor and antibiotic resistance genes (PastyVRDB) allowing for detailed characterization of isolates. The majority of human P. multocida isolates encoded a reduced range of iron receptors and contained only one filamentous hemagglutinin gene. Finally, gene-trait analysis identified a putative L-fucose uptake and utilization pathway that was over-represented in subsp. septica strains and may represent a novel host predilection mechanism in this subspecies. Together, these analyses have identified pathogenic mechanisms likely important for P. multocida zoonotic infections.IMPORTANCEPasteurella multocida can cause serious infections in humans, including skin and wound infections, pneumonia, peritonitis, meningitis, and bacteraemia. Cats and dogs are known vectors of human pasteurellosis, transmitting P. multocida via bite wounds or contact with animal saliva. The mechanisms that underpin P. multocida human predilection and pathogenesis are poorly understood. With increasing identification of antibiotic-resistant P. multocida strains, understanding these mechanisms is vital for developing novel treatments and control strategies to combat P. multocida human infection. Here, we show that a narrow range of P. multocida strains cause disease in humans, while cats and dogs, common vectors for zoonotic infections, can harbor a wide range of P. multocida strains. We also present a curated P. multocida-specific database, allowing quick and detailed characterization of newly sequenced P. multocida isolates.

Ceftolozane-Tazobactam against Pseudomonas aeruginosa Cystic Fibrosis Clinical Isolates in the Hollow-Fiber Infection Model: Challenges Imposed by Hypermutability and Heteroresistance.

Pseudomonas aeruginosa remains a challenge in chronic respiratory infections in cystic fibrosis (CF). Ceftolozane-tazobactam has not yet been evaluated against multidrug-resistant hypermutable P. aeruginosa isolates in the hollow-fiber infection model (HFIM). Isolates CW41, CW35, and CW44 (ceftolozane-tazobactam MICs of 4, 4, and 2 mg/L, respectively) from adults with CF were exposed to simulated representative epithelial lining fluid pharmacokinetics of ceftolozane-tazobactam in the HFIM. Regimens were continuous infusion (CI; 4.5 g/day to 9 g/day, all isolates) and 1-h infusions (1.5 g every 8 hours and 3 g every 8 hours, CW41). Whole-genome sequencing and mechanism-based modeling were performed for CW41. CW41 (in four of five biological replicates) and CW44 harbored preexisting resistant subpopulations; CW35 did not. For replicates 1 to 4 of CW41 and CW44, 9 g/day CI decreased bacterial counts to <3 log10 CFU/mL for 24 to 48 h, followed by regrowth and resistance amplification. Replicate 5 of CW41 had no preexisting subpopulations and was suppressed below ~3 log10 CFU/mL for 120 h by 9 g/day CI, followed by resistant regrowth. Both CI regimens reduced CW35 bacterial counts to <1 log10 CFU/mL by 120 h without regrowth. These results corresponded with the presence or absence of preexisting resistant subpopulations and resistance-associated mutations at baseline. Mutations in ampC, algO, and mexY were identified following CW41 exposure to ceftolozane-tazobactam at 167 to 215 h. Mechanism-based modeling well described total and resistant bacterial counts. The findings highlight the impact of heteroresistance and baseline mutations on the effect of ceftolozane-tazobactam and limitations of MIC to predict bacterial outcomes. The resistance amplification in two of three isolates supports current guidelines that ceftolozane-tazobactam should be utilized together with another antibiotic against P. aeruginosa in CF.

Genome-Wide Investigation of Pasteurella multocida Identifies the Stringent Response as a Negative Regulator of Hyaluronic Acid Capsule Production.

Pasteurella multocida is a Gram-negative capsulated bacterium responsible for a range of diseases that cause severe morbidity and mortality in livestock animals. The hyaluronic acid (HA) capsule produced by P. multocida serogroup A strains is a critical virulence factor. In this study, we utilized transposon-directed insertion site sequencing (TraDIS) to identify genes essential for in vitro growth of P. multocida and combined TraDIS with discontinuous density gradients (TraDISort) to identify genes required for HA capsule production and regulation in this pathogen. Analysis of mutants with a high cell density phenotype, indicative of the loss of extracellular capsule, led to the identification of 69 genes important for capsule production. These genes included all previously characterized genes in the capsule biosynthesis locus and fis and hfq, which encode known positive regulators of P. multocida capsule. Many of the other capsule-associated genes identified in this study were involved in regulation or activation of the stringent response, including spoT and relA, which encode proteins that regulate the concentration of guanosine alarmones. Disruption of the autoregulatory domains in the C-terminal half of SpoT using insertional mutagenesis resulted in reduced expression of capsule biosynthesis genes and an acapsular phenotype. Overall, these findings have greatly increased the understanding of hyaluronic acid capsule production and regulation in P. multocida. IMPORTANCE The bacterial pathogen P. multocida can cause serious disease in production animals, including fowl cholera in poultry, hemorrhagic septicemia in cattle and buffalo, atrophic rhinitis in pigs, and respiratory diseases in a range of livestock. P. multocida produces a capsule that is essential for systemic disease, but the complete mechanisms underlying synthesis and regulation of capsule production are not fully elucidated. A whole-genome analysis using TraDIS was undertaken to identify genes essential for growth in rich media and to obtain a comprehensive characterization of capsule production. Many of the capsule-associated genes identified in this study were involved in the stringent response to stress, a novel finding for this important animal pathogen.

Does economic optimisation explain LAI and leaf trait distributions across an Amazon soil moisture gradient?

Leaf area index (LAI) underpins terrestrial ecosystem functioning, yet our ability to predict LAI remains limited. Across Amazon forests, mean LAI, LAI seasonal dynamics and leaf traits vary with soil moisture stress. We hypothesise that LAI variation can be predicted via an optimality-based approach, using net canopy C export (NCE, photosynthesis minus the C cost of leaf growth and maintenance) as a fitness proxy. We applied a process-based terrestrial ecosystem model to seven plots across a moisture stress gradient with detailed in situ measurements, to determine nominal plant C budgets. For each plot, we then compared observations and simulations of the nominal (i.e. observed) C budget to simulations of alternative, experimental budgets. Experimental budgets were generated by forcing the model with synthetic LAI timeseries (across a range of mean LAI and LAI seasonality) and different leaf trait combinations (leaf mass per unit area, lifespan, photosynthetic capacity and respiration rate) operating along the leaf economic spectrum. Observed mean LAI and LAI seasonality across the soil moisture stress gradient maximised NCE, and were therefore consistent with optimality-based predictions. Yet, the predictive power of an optimality-based approach was limited due to the asymptotic response of simulated NCE to mean LAI and LAI seasonality. Leaf traits fundamentally shaped the C budget, determining simulated optimal LAI and total NCE. Long-lived leaves with lower maximum photosynthetic capacity maximised simulated NCE under aseasonal high mean LAI, with the reverse found for short-lived leaves and higher maximum photosynthetic capacity. The simulated leaf trait LAI trade-offs were consistent with observed distributions. We suggest that a range of LAI strategies could be equally economically viable at local level, though we note several ecological limitations to this interpretation (e.g. between-plant competition). In addition, we show how leaf trait trade-offs enable divergence in canopy strategies. Our results also allow an assessment of the usefulness of optimality-based approaches in simulating primary tropical forest functioning, evaluated against in situ data.

The biomechanical and morphological characteristics of the ligamentum mucosum and its potential role in anterior knee pain.

BACKGROUND: The ligamentum mucosum is composed of dense regular connective tissue and traverses from the distal femur to the infrapatellar fat pad. While the gross and histologic morphology has been studied, there is currently no evidence concerning the biomechanical properties of the ligamentum mucosum and the potential of anterior knee pain. The purpose of this study was to determine the anatomical, mechanical and histological properties of the ligamentum mucosum. METHODS: Dissections were performed on cadaveric knee specimens (N = 18) and histological analysis (n = 6) was performed to define the anatomical characteristics of the ligamentum mucosum using standard hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical methods. Biomechanical testing (n = 5) was conducted to determine the tensile properties of the ligamentum mucosum. The peak load at failure, stiffness, and strain were analyzed. RESULTS: Sixty-four percent of the knees had a ligamentum mucosum and the histological analysis confirmed it to be composed of dense regular connective tissue. Small peripheral nerves were identified in the junction between the ligamentum mucosum and the fat pad. The average (SD) peak force of failure, stiffness, and strain were 31.9 N (19.0), 5.1 N/mm (3.59), and 0.83 (0.14), respectively. CONCLUSIONS: The tensile strength and stiffness of the ligamentum mucosum is considerably less than the primary stabilizers of the knee joint. Based on these findings, it is improbable that the ligamentum mucosum has a meaningful effect on the kinematics of the extensor mechanism; perturbations of the tissue and the connected infrapatellar fat pad could potentially play a role in the pathogenesis of anterior knee pain.

Arthroscopic Untethering of the Fat Pad of the Knee: Release or Resection of the Infrapatellar Plica (Ligamentum Mucosum) and Related Structures for Anterior Knee Pain.

Anterior knee pain (AKP), a multifactorial symptom complex, can be successfully treated surgically. A specific diagnosis often cannot be made, but the pain is linked to an unrecognized common factor in most patients: the mechanical behavior of the non-isometric contents of the anterior compartment of the knee-the fat pad (FP) and infrapatellar plica (IPP). The objective of this presentation is to describe an effective arthroscopic technique that treats AKP by addressing this common factor. The operation consists of release or resection of the IPP, or ligamentum mucosum, which tethers the FP. These highly innervated tissues act together as a hydraulic shock absorber, filling the anterior compartment. They stretch and deform at the extremes of knee motion because of constraint centrally by the non-isometric IPP. These dynamic changes in shape are eliminated when the plica is released or resected. Pain perception is from perturbed nociceptive nerves: pain relief results from de-tensioning these contained nerves by untethering the fat pad. Ascribing pain causation is problematic because morphologic change, such as inflammation, fibrosis, or contracture of these structures, is only present in a minority of cases. Nonetheless, AKP is both physically linked to these central, pain-sensitive structures and relieved by this operation.