Detection of critical cerebral desaturation thresholds by three regional oximeters during hypoxia: a pilot study in healthy volunteers.

BACKGROUND: Regional oximetry is increasingly used to monitor post-extraction oxygen status of the brain during surgical procedures where hemodynamic fluctuations are expected. Particularly in cardiac surgery, clinicians employ an interventional algorithm to restore baseline regional oxygen saturation (rSO2) when a patient reaches a critical desaturation threshold. Evidence suggests that monitoring cardiac surgery patients and intervening to maintain rSO2 can improve postoperative outcomes; however, evidence generated with one manufacturer's device may not be applicable to others. We hypothesized that regional oximeters from different manufacturers respond uniquely to changes in oxygen saturation in healthy volunteers. METHODS: Three devices were tested: INVOS™ 5100C (Medtronic), EQUANOX™ 7600 (Nonin), and FORE-SIGHT™ (CASMED) monitors. We divided ten healthy subjects into two cohorts wearing a single sensor each from INVOS and EQUANOX (n = 6), or INVOS and FORE-SIGHT (n = 4). We induced and reversed hypoxia by adjusting the fraction of inspired oxygen. We calculated the magnitude of absolute rSO2 change and rate of rSO2 change during desaturation and resaturation, and determined if and when each device reached a critical interventional rSO2 threshold during hypoxia. RESULTS: All devices responded to changes in oxygen directionally as expected. The median absolute rSO2 change and the rate of rSO2 change was significantly greater during desaturation and resaturation for INVOS compared with EQUANOX (P = 0.04). A similar but nonsignificant trend was observed for INVOS compared with FORE-SIGHT; our study was underpowered to definitively conclude there was no difference. A 10% relative decrease in rSO2 during desaturation was detected by all three devices across the ten subjects. INVOS met a 20% relative decrease threshold in all subjects of both cohorts, compared to 1 with EQUANOX and 2 with FORE-SIGHT. Neither EQUANOX nor FORE-SIGHT reached a 50% absolute rSO2 threshold compared with 4 and 3 subjects in each cohort with INVOS, respectively. CONCLUSIONS: Significant differences exist between the devices in how they respond to changes in oxygen saturation in healthy volunteers. We suggest caution when applying evidence generated with one manufacturer's device to all devices.

Effects of the twin-arginine translocase on the structure and antimicrobial susceptibility of Escherichia coli biofilms.

In this descriptive study, we used Escherichia coli twin-arginine translocase (tat) mutants to distinguish antibiotic tolerance from the formation of mature biofilm structure. Biofilm formation by wild-type and deltatat strains of E. coli was evaluated using viable cell counts, scanning electron microscopy, and confocal laser-scanning microscopy. Escherichia coli deltatat mutants had an impaired ability to form biofilms when grown in rich or minimal media. These mutants produced disorganized layers and cell aggregates with significantly decreased cell density relative to the wild-type strain. In contrast, wild-type E. coli grown under similar test conditions formed highly structured, surface-adherent communities. We thus determined if this decreased biofilm formation by E. coli deltatat mutants may result in lowered tolerance to antimicrobials. When grown in rich media, planktonic deltatat mutants were hypersensitive to some metals, detergents, and antibiotics. However, the corresponding biofilms were about as resilient as the wild-type strain. In contrast, both planktonic cells and biofilms of the deltatatABC strain grown in minimal media were hypersensitive to many antimicrobials. Remarkably, these biofilms remained up to 365 times more tolerant to beta-lactams than corresponding planktonic cells. Our data suggest that the twin-arginine translocase may play a contributing role in the antimicrobial tolerance, structural organization, and formation of mature E. coli biofilms under nutrient-limited conditions. However, the high tolerance of the deltatatABC strain to bactericidal concentrations of antimicrobials indicates that mature biofilm structure may not be required for surface-adherent E. coli to survive exposure to these lethal factors.

Quorum-sensing mutations affect attachment and stability of Burkholderia cenocepacia biofilms.

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.

Enhanced Pseudomonas aeruginosa biofilm development mediated by human neutrophils.

Cystic fibrosis (CF) lung disease features persistent neutrophil accumulation to the airways from the time of infancy. CF children are frequently exposed to Pseudomonas aeruginosa, and by adulthood, 80% of CF patients are chronically infected. The formation of biofilms is a particularly important phenotypic characteristic of P. aeruginosa that allows for bacterial survival despite aggressive antibiotic therapy and an exuberant immune response. Here, we show that the presence of neutrophils enhances initial P. aeruginosa biofilm development over a period of 72 h through the formation of polymers comprised of actin and DNA. F-actin was found to be a site of attachment for P. aeruginosa. These actin and DNA polymers are present in CF sputum, and disruption of the polymers dispersed the associated P. aeruginosa cells and reduced biofilm development. These findings demonstrate a potential maladaptation of the primary innate response. When the host fails to eradicate the infection, cellular components from necrotic neutrophils can serve as a biological matrix to facilitate P. aeruginosa biofilm formation.

Green and red fluorescent protein vectors for use in biofilm studies of the intrinsically resistant Burkholderia cepacia complex.

Cystic fibrosis isolates of the Burkholderia cepacia complex (BCC) have demonstrated a propensity to associate intimately with Pseudomonas aeruginosa in mixed community biofilms, which may impact on their overall pathogenicity during infection of the lungs in cystic fibrosis. Here, we describe the construction and use of novel green and red fluorescent protein expression vectors suitable for labeling biofilm cells of multi-resistant clinical isolates of the BCC for microscopic analysis of both single species biofilms and mixed community associations with P. aeruginosa. Antimicrobial susceptibility testing established that tetracycline and/or trimethoprim were suitable selective agents for widespread use in BCC. The green and red fluorescent protein genes, driven by constitutively active promoters, were cloned into two mobilizable plasmids pBBR1MCS-3 and pBBR1Tp, carrying tetracycline and trimethoprim resistance cassettes, respectively. The fluorescence of transformed BCC and P. aeruginosa planktonic cells was detectable using fluorescence microscopy and/or fluorometry. The plasmids were stable in the absence of selection for at least 3 days in planktonic and biofilm cultures, and fluorescence was still visible in a 4-day glass coverslip flow cell biofilm. The plasmids functioned well to distinguish the two species in a mixed community biofilm, with no indications of plasmid transfer between species or cross-talk of the fluorescent signals. These vectors represent the first green and red fluorescent vectors to be constructed and analyzed specifically for wide spread use in BCC and P. aeruginosa single and mixed biofilm cultures.