Bacterial aerobic respiration is a major consumer of oxygen in sputum from patients with acute lower respiratory tract infection.

Bacterial aerobic respiration may determine the outcome of antibiotic treatment in experimental settings, but the clinical relevance of bacterial aerobic respiration for the outcome of antibiotic treatment has not been tested. Therefore, we hypothesized that bacterial aerobic respiration is higher in sputum from patients with acute lower respiratory tract infections (aLRTI), than in sputum from patients with chronic LRTI (cLRTI), where the bacteria persist despite antibiotic treatment. The bacterial aerobic respiration was determined according to the dynamics of the oxygen (O2 ) concentration in sputum from aLRTI patients (n = 52). This result was evaluated by comparison to previously published data from patients with cLRTI. O2 consumption resulting in anoxic zones was more frequent in sputum with detected bacterial pathogens. The bacterial aerobic respiration in aLRTI sputum approximated 55% of the total O2 consumption, which was significantly higher than previously published for cLRTI. The bacterial aerobic respiration in sputum was higher in aLRTI patients than previously seen in cLRTI patients, indicating the presence of bacteria with a sensitive physiology in aLRTI. These variations in bacterial physiology between aLRTI patients and cLRTI patients may contribute the huge difference in treatment success between the two patient groups.

Exhaled nitric oxide in intubated ICU patients on mechanical ventilation-a feasibility study.

It can be a clinical challenge to distinguish inflammation from infection in critically ill patients. Therefore, valid and conclusive surrogate markers for infections are desired. Nitric oxide (NO) might be that marker since concentrations of exhaled NO have shown to change in the presence of various diseases. This observational, prospective, single-center feasibility study aimed to investigate if fractional exhaled NO (FeNO) can be measured in intubated patients with or without infection, pneumonia and septic shock in a standardized, reliable setting. 20 intubated patients in the intensive care unit (ICU) were included for analysis. FeNO mean values were measured in the endotracheal tube via the suction channel using a chemiluminescence based analyzer. We developed a pragmatic method to measure FeNO repeatedly and reliably in intubated patients using a chemiluminescence based analyzer. We found a median of 0.98 (0.59-1.44) FeNO mean (ppb) in exhaled breath from all 20 intubated patient. Intubated patient with suspected infection had a significantly lower median FeNO mean compared with the intubated patients without suspected infection. Similarly did patients with septic shock demonstrate a significantly lower median FeNO mean than without septic shock. We found no statistical difference in median FeNO mean for intubated patients with pneumonia. It was feasible to measure FeNO in intubated patients in the ICU. Our results indicate decreased levels of FeNO in infected intubated patients in the ICU. The study was not powered to provide firm conclusions, so larger trials are needed to confirm the results and to prove FeNO as a useful biomarker for distinguishment between infection and inflammation in the ICU.

Antimicrobial resistance of Pseudomonas aeruginosa in a cystic fibrosis population after introduction of a novel cephalosporin/ß-lactamase inhibitor combination.

Ceftolozane-tazobactam is a new ß-lactam/ß-lactamase inhibitor combination approved by the U.S. Food and Drug Administration in 2019 for the treatment of hospital-acquired and ventilator-associated pneumonia. The combination is a particularly potent inhibitor of penicillin-binding proteins with higher affinity than other ß-lactam agents. Persons with cystic fibrosis (pwCF) often harbour resistant Gram-negative bacteria in the airways and need antibiotics to prevent declining lung function. To test whether the introduction of ceftolozane-tazobactam in the period 2015-2020 led to a bacterial population level increase in cephalosporin resistance in a Danish CF population. In vitro, activity of ceftolozane-tazobactam was evaluated by susceptibility testing of clinical Pseudomonas aeruginosa isolated from pwCF from January 1, 2015, to June 1, 2020. Six thousand three hundred thirty two isolates collected from 210 adult pwCF were included. Thirty pwCF were treated with ceftolozane-tazobactam at least once. Ceftolozane-tazobactam exposure did not increase cephalosporin resistance on an individual or population level. However, resistance to ceftolozane-tazobactam was recorded despite no prior exposure in four pwCF. Compared to ceftazidime, ceftolozane-tazobactam had a better in vitro activity on P. aeruginosa. The percentage of non-mucoid P. aeruginosa isolates susceptible to ceftolozane-tazobactam were higher or equal to 5 other ß-lactams. Ceftolozane-tazobactam expands the armamentaria against P. aeruginosa with acceptable levels for a selection of drug resistance.

Adaptation of Pseudomonas aeruginosa biofilms to tobramycin and the quorum sensing inhibitor C-30 during experimental evolution requires multiple genotypic and phenotypic changes.

In the present study we evaluated the fitness, antimicrobial susceptibility, metabolic activity, gene expression, in vitro production of virulence factors and in vivo virulence of experimentally evolved Pseudomonas aeruginosa PAO1. These strains were previously evolved in the presence of tobramycin and the quorum sensing inhibitor furanone C-30 (C-30) and carried mutations in mexT and fusA1. Compared to the wild-type (WT), the evolved strains show a different growth rate and different metabolic activity, suggesting they have an altered fitness. mexT mutants were less susceptible to C-30 than WT strains; they also show reduced susceptibility to chloramphenicol and ciprofloxacin, two substrates of the MexEF-OprN efflux pump. fusA1 mutants had a decreased susceptibility to aminoglycoside antibiotics, and an increased susceptibility to chloramphenicol. The decreased antimicrobial susceptibility and decreased susceptibility to C-30 was accompanied by a changed metabolic activity profile during treatment. The expression of mexE was significantly increased in mexT mutants and induced by C-30, suggesting that MexEF-OprN exports C-30 out of the bacterial cell. The in vitro production of virulence factors as well as virulence in two in vivo models of the strains evolved in the presence of C-30 was unchanged compared to the virulence of the WT. Finally, the evolved strains were less susceptible towards tobramycin (alone and combined with C-30) in an in vivo mouse model. In conclusion, this study shows that mutations acquired during experimental evolution of P. aeruginosa biofilms in the presence of tobramycin and C-30, are accompanied by an altered fitness, metabolism, mexE expression and in vitro and in vivo antimicrobial susceptibility.

Hyperbaric oxygen therapy augments ciprofloxacin effect against Pseudomonas aeruginosa biofilm infected chronic wounds in a mouse model.

Introduction: Chronic wounds have a compromised microcirculation which leads to restricted gas exchange. The majority of these hypoxic wounds is infested with microorganisms congregating in biofilms which further hinders the antibiotic function. We speculate whether this process can be counteracted by hyperbaric oxygen therapy (HBOT). Methodology: Twenty-eight BALB/c mice with third-degree burns were included in the analyses. Pseudomonas aeruginosa embedded in seaweed alginate beads was injected under the eschar to mimic a biofilm infected wound. Challenged mice were randomized to receive either 4 days with 1 x ciprofloxacin combined with 2 × 90 min HBOT at 2.8 standard atmosphere daily, 1 x ciprofloxacin as monotherapy or saline as placebo. The mice were clinically scored, and wound sizes were estimated by planimetry daily. Euthanasia was performed on day 8. Wounds were surgically removed in toto, homogenized and plated for quantitative bacteriology. Homogenate supernatants were used for cytokine analysis. Results: P. aeruginosa was present in all wounds at euthanasia. A significant lower bacterial load was seen in the HBOT group compared to either the monotherapy ciprofloxacin group (p = 0.0008), or the placebo group (p < 0.0001). IL-1ß level was significantly lower in the HBOT group compared to the placebo group (p = 0.0007). Both treatment groups had higher osteopontin levels than the placebo group (p = 0.002 and p = 0.004). The same pattern was seen in the S100A9 analysis (p = 0.01 and p = 0.008), whereas no differences were detected between the S100A8, the VEGF or the MMP8 levels in the three groups. Conclusion: These findings show that HBOT improves the bactericidal activity of ciprofloxacin against P. aeruginosa wound biofilm in vivo. HBOT in addition to ciprofloxacin also modulates the host response to a less inflammatory phenotype.

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.

Prevalence of biofilms in acute infections challenges a longstanding paradigm.

The significance of bacterial biofilm formation in chronic bacterial lung infections has long been recognized [1]. Likewise, chronic biofilm formation on medical devices is well accepted as a nidus for recurrent bacteremia [2,3]. Even though the prevailing paradigm relies on the dominance of planktonic bacteria in acute endobronchial infections, our understanding of the bacterial organization during acute infection is, so far, limited - virtually absent. However, by comparing similar clinical samples, we have recently demonstrated massive bacterial biofilm formation during acute lung infections resembling the immense bacterial biofilm formation during chronic lung infections. These findings pose major challenges to the basic paradigm of chronic infections being dominated by biofilm forming bacteria while acute infections are dominated by planktonic bacteria. As opposed to the similar high amount of bacterial biofilm found in chronic and acute lung infections, we found that the fast bacterial growth in acute lung infections differed from the slow bacterial growth in chronic lung infections. By highlighting these new findings, we review modes of improved treatment of biofilm infections and the relevance of bacterial growth rates for other bacterial biofilm infections than human lung infections.

Increased sputum lactate during oral glucose tolerance test in cystic fibrosis.

Blood glucose levels exceeding 8 mM are shown to increase glucose levels in airway surface in cystic fibrosis (CF). Moreover, high levels of endobronchial glucose are proposed to increase the growth of common CF bacteria and feed the neutrophil-driven inflammation. In the infected airways, glucose may be metabolized by glycolysis to lactate by both bacteria and neutrophils. Therefore, we aimed to investigate whether increased blood glucose may fuel the glycolytic pathways of the lung inflammation by determining sputum glucose and lactate during an oral glucose tolerance test (OGTT). Sputum from 27 CF patients was collected during an OGTT. Sputum was collected at fasting and one and two hours following the intake of 75 g of glucose. Only participants able to expectorate more than one sputum sample were included. Glucose levels in venous blood and lactate and glucose content in sputum were analyzed using a regular blood gas analyzer. We collected 62 sputum samples: 20 at baseline, 22 after 1 h, and 20 after 2 h. Lactate and glucose were detectable in 30 (48.4%) and 43 (69.4%) sputum samples, respectively. The sputum lactate increased significantly at 2 h in the OGTT (p = 0.024), but sputum glucose was not changed. As expected, plasma glucose level significantly increased during the OGTT (p < 0.001). In CF patients, sputum lactate increased during an OGTT, while the sputum glucose did not reflect the increased plasma glucose. The increase in sputum lactate suggests that glucose spills over from plasma to sputum where glucose may enhance the inflammation by fueling the anaerobic metabolism in neutrophils or bacteria.

Adaptive Immune Response to Mycobacterium abscessus Complex (MABSC) in Cystic Fibrosis and the Implications of Cross-Reactivity.

Background: We aimed to characterise the adaptive immune response to Mycobacterium abscessus complex (MABSC) and its cross-reactivity with Mycobacterium avium complex (MAC) and Mycobacterium bovis (Bacille Calmette-Guérin, BCG) in cystic fibrosis (CF) patients and non-CF controls in terms of lymphocyte proliferation and immunophenotyping, cytokine production and anti-MABSC IgG plasma levels. Methods: In this cross-sectional analysis, peripheral blood mononuclear cells (PBMC) from CF patients with MABSC (CF/MABSC, n=12), MAC infection history (CF/MAC, n=5), no NTM history (CF/NTM-, n=15), BCG-vaccinated (C/BCG+, n=9) and non-vaccinated controls (C/BCG-, n=8) were cultured for four days under stimulation with an in-house MABSC lysate and we used flow cytometry to assess lymphocyte proliferation (given by lymphoblast formation) and immunophenotypes. Cytokine production was assessed after overnight whole blood stimulation with the same lysate, and anti-MABSC IgG levels were measured in plasma from non-stimulated blood. Results: All CF/MABSC patients had increased CD3+ and CD19+ lymphoblast formation upon PBMC stimulation with MABSC lysate. There was a higher rate of CD3+ than CD19+ lymphoblasts, predominance of CD4+ over CD8+ lymphoblasts, IFN-γ, TNF-α and IL-2 production, low production of the Th17-associated IL-17, and discrete or no production of Th2/B cell-associated cytokines soluble CD40 ligand (CD40L), IL-4 and IL-5, indicating a Th1-dominated phenotype and infection restricted to the lungs. A similar pattern was seen in C/BCG+ controls, and CF/MAC patients, pointing to cross-reactivity. MABSC-IgG levels were higher in CF/MABSC patients than in both control groups, but not CF/NTM- patients, most of whom also had CD3+ and/or CD19+ lymphoblast formation upon PBMC stimulation, indicating previous exposure, subclinical or latent infection with MABSC or other NTM. Conclusion: The anti-MABSC immune response is Th1-skewed and underlines the cross-reactivity in the anti-mycobacterial immune response. The results, together with published clinical observations, indicate that BCG vaccination may cross-react against NTM in CF patients, and this should be investigated. Due to cross-reactivity, it would also be interesting to investigate whether a combination of MABSC-induced cytokine production by blood cells and anti-MABSC IgG measurement can be useful for identifying latent or subclinical infection both with MABSC and other NTM in CF patients.

The structure-function relationship of Pseudomonas aeruginosa in infections and its influence on the microenvironment.

Pseudomonas aeruginosa is a human pathogen associated with both acute and chronic infections. While intensively studied, the basic mechanisms enabling the long-term survival of P. aeruginosa in the host, despite massive immune system attack and heavy antimicrobial treatment, remain to be identified. We argue that such infections may represent niche invasions by P. aeruginosa that influence the microenvironment by depleting host-derived substrate and activating the immune response. Bacteria embedded in cell aggregates establish a microenvironmental niche, where they endure the initial host response by slowing down their metabolism. This provides stable, lasting growth conditions with a constant, albeit slow supply of substrate and electron acceptors. Under such stable conditions, P. aeruginosa exhibits distinct adaptive traits, where its gene expression pattern reflects a life exposed to continuous attack by the host immune system and antimicrobials. Here, we review fundamental microenvironmental aspects of chronic P. aeruginosa infections and examine how their structural organization influences their in vivo microenvironment, which in turn affects the interaction of P. aeruginosa biofilm aggregates with the host immune system. We discuss how improving our knowledge about the microenvironmental ecology of P. aeruginosa in chronic infections can be used to combat persistent, hard-to-treat bacterial infections.

Endotracheal lactate reflects lower respiratory tract infections and inflammation in intubated patients.

The aim of this study was to assess L-lactate and D-lactate in endotracheal aspirate from intubated patients hospitalized at the intensive care unit and explore their use as diagnostic biomarkers for inflammation and lower respiratory tract infections (LRTI). Tracheal aspirates from 91 intubated patients were obtained at time of intubation and sent for microbiological analyses, neutrophil count, and colorimetric lactate measurements. We compared the concentration of lactate from patients with microbiological verified LRTI or clinical/radiological suspicion of LRTI with a control group. In addition, associations between inflammation and the lactate isomers were examined by correlating L-lactate and D-lactate with sputum neutrophils and clinical assessments. The concentration of L-lactate was increased in aspirates with verified or suspected LRTI (p < 0.001) relative to the control group at Day 0. Connections between L-lactate and inflammation were indicated by the correlation between neutrophils and L-lactate (p < 0.001). We found no increase in sputum D-lactate from patients with verified or suspected LRTI relative to the control group and D-lactate was not correlated with neutrophils. L-lactate was found to be a potential indicator for inflammation and LRTI at the time of intubation. An association was found between neutrophil count and L-lactate. Interestingly, the increase of L-lactate in the control group after intubation may suggest that intubation challenges the host response by inflicting tissue damage or by introducing infectious microbes.

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.

Nitric-oxide-driven oxygen release in anoxic Pseudomonas aeruginosa.

Denitrification supports anoxic growth of Pseudomonas aeruginosa in infections. Moreover, denitrification may provide oxygen (O2) resulting from dismutation of the denitrification intermediate nitric oxide (NO) as seen in Methylomirabilis oxyfera. To examine the prevalence of NO dismutation we studied O2 release by P. aeruginosa in airtight vials. P. aeruginosa rapidly depleted O2 but NO supplementation generated peaks of O2 at the onset of anoxia, and we demonstrate a direct role of NO in the O2 release. However, we were not able to detect genetic evidence for putative NO dismutases. The supply of endogenous O2 at the onset of anoxia could play an adaptive role when P. aeruginosa enters anaerobiosis. Furthermore, O2 generation by NO dismutation may be more widespread than indicated by the reports on the distribution of homologues genes. In general, NO dismutation may allow removal of nitrate by denitrification without release of the very potent greenhouse gas, nitrous oxide.

Immune Responses to Pseudomonas aeruginosa Biofilm Infections.

Pseudomonas aeruginosa is a key pathogen of chronic infections in the lungs of cystic fibrosis patients and in patients suffering from chronic wounds of diverse etiology. In these infections the bacteria congregate in biofilms and cannot be eradicated by standard antibiotic treatment or host immune responses. The persistent biofilms induce a hyper inflammatory state that results in collateral damage of the adjacent host tissue. The host fails to eradicate the biofilm infection, resulting in hindered remodeling and healing. In the present review we describe our current understanding of innate and adaptive immune responses elicited by P. aeruginosa biofilms in cystic fibrosis lung infections and chronic wounds. This includes the mechanisms that are involved in the activation of the immune responses, as well as the effector functions, the antimicrobial components and the associated tissue destruction. The mechanisms by which the biofilms evade immune responses, and potential treatment targets of the immune response are also discussed.

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.

Neutrophil count in sputum is associated with increased sputum glucose and sputum L-lactate in cystic fibrosis.

BACKGROUND: Markers of lung inflammation measured directly in expectorated sputum have the potential of improving the timing of antibiotic treatment in cystic fibrosis (CF). L-Lactate might be a marker of inflammation, as it is produced from glucose by polymorphonuclear neutrophils (PMNs) in CF lungs. We aimed to investigate changes in and associations between PMNs, glucose and L-lactate in sputum during antibiotic treatment. In addition, the effect of hemoglobin A1c and plasma glucose on these biomarkers were investigated. METHODS: We sampled non-induced sputum at day 0, 7, 14 and 42 in 27 chronically infected CF patients electively treated with 14 days of intravenous antibiotic. To analyze sputum samples, we used flowcytometry to count PMNs and colorimetric assays to estimate lactate and glucose. RESULTS: No changes in levels of PMNs, glucose and lactate were detected in sputum during the antibiotic treatment. Sputum PMNs were positively associated with both glucose (log coefficient = 0.20, p = 0.01) and L-lactate (log coefficient = 0.34, p<0.001). In multivariate analyses, hemoglobin A1c was negatively associated with sputum PMNs (log coefficient = -1.68, p<0.001) and plasma glucose was negatively associated with sputum glucose (log coefficient = -0.09, p = 0.02). CONCLUSIONS: In CF sputum PMNs, glucose and lactate were unchanged during elective antibiotic treatment. However, sputum PMNs were associated with both sputum glucose and sputum lactate. Surprisingly, hyperglycemia seemed to be associated with less PMNs infiltration and less glucose in CF sputum.

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.

Oxygen Restriction Generates Difficult-to-Culture P. aeruginosa.

Induction of a non-culturable state has been demonstrated for many bacteria, e.g., Escherichia coli and various Vibrio spp. In a clinical perspective, the lack of growth due to these non-culturable bacteria can have major consequences for the treatment of patients. Here, we show how anoxic conditioning (restriction of molecular oxygen, O2) generates difficult-to-culture (DTC) bacteria during biofilm growth. A significant subpopulation of Pseudomonas aeruginosa entered a DTC state after anoxic conditioning, ranging from 5 to 90% of the total culturable population, in both planktonic and biofilm models. Anoxic conditioning also generated DTC subpopulations of Staphylococcus aureus and Staphylococcus epidermidis (89 and 42% of the total culturable population, respectively). Growth of the DTC populations were achieved by substituting O2 with 10 mM NO3 - as an alternative electron acceptor for anaerobic respiration or, in the case of P. aeruginosa, by adding sodium pyruvate or catalase as scavengers against reactive oxygen species (ROS) during aerobic respiration. An increase in normoxic plating due to addition of catalase suggests the molecule hydrogen peroxide as a possible mechanism for induction of DTC P. aeruginosa. Anoxic conditioning also generated a true viable but non-culturable (VBNC) population of P. aeruginosa that was not resurrected by substituting O2 with NO3 - during anaerobic respiration. These results demonstrate that habituation to an anoxic micro-environment could complicate diagnostic culturing of bacteria, especially in the case of chronic infections where oxygen is restricted due to the host immune response.

Hyperbaric oxygen treatment increases killing of aggregating Pseudomonas aeruginosa isolates from cystic fibrosis patients.

BACKGROUND: Pseudomonas aeruginosa is a major pathogen of the chronic lung infections in cystic fibrosis (CF) patients. These persistent bacterial infections are characterized by bacterial aggregates with biofilm-like properties and are treated with nebulized or intravenous tobramycin in combination with other antibiotics. However, the chronic infections are close to impossible to eradicate due to reasons that are far from fully understood. Recent work has shown that re­oxygenation of hypoxic aggregates by hyperbaric oxygen (O2) treatment (HBOT: 100% O2 at 2.8 bar) will increase killing of aggregating bacteria by antibiotics. This is relevant for treatment of infected CF patients where bacterial aggregates are found in the endobronchial secretions that are depleted of O2 by the metabolism of polymorphonuclear leukocytes (PMNs). The main objective of this study was to investigate the effect of HBOT as an adjuvant to tobramycin treatment of aggregates formed by P. aeruginosa isolates from CF patients. METHODS: The effect was tested using a model with bacterial aggregates embedded in agarose. O2 profiling was used to confirm re­oxygenation of aggregates. RESULTS: We found that HBOT was able to significantly enhance the effect of tobramycin against aggregates of all the P. aeruginosa isolates in vitro. The effect was attributed to increased O2 levels leading to increased growth and thus increased uptake of and killing by tobramycin. CONCLUSIONS: Re­oxygenation may in the future be a clinical possibility as adjuvant to enhance killing by antibiotics in cystic fibrosis lung infections.

The inflamed sputum in lower respiratory tract infection: l-lactate levels are correlated to neutrophil accumulation.

Lower respiratory tract infections (LRTI) are common, but little is known about the response of biomarkers of inflammation in the lungs. Therefore, our primary aim was to compare the concentration of l-lactate to the concentration of neutrophils in sputum and systemic markers of infection. Because it is difficult to differentiate viral and bacterial infection on the basis of clinical presentation in LRTI, our secondary aim was to evaluate if l- and d-lactate may serve as markers of local inflammation as representatives of neutrophils and bacteria, respectively. METHODS: Patients with acute LRTI were prospectively recruited. Sputum samples were collected and analysed for neutrophil count, l-lactate and d-lactate. We had data on pathogens from sputum cultures and polymerase chain reaction (PCR) (atypical bacteria, virus) and C-reactive protein (CRP) from blood. RESULTS: In 44 sputum samples from 32 patients, the median (interquartile range (IQR)) sputum neutrophil granulocyte count was 0.615 × 107  cells/mL (0.236-1.575). The sputum neutrophil granulocyte count was associated with sputum l-lactate (p = 0.011) and CRP (p = 0.018), but not with d-lactate (p = 0.177). There was a strong association between sputum d-lactate and l-lactate (p < 0.0001). CONCLUSION: As l-lactate in sputum is closely correlated to sequestration of neutrophils in the lungs, l-lactate is a marker for local inflammation in LRTI and a potential biomarker in clinical management of LRTI. On expectorated sputum, d-lactate had no clinical relevance.