Environmentally Endemic Pseudomonas aeruginosa Strains with Mutations in lasR Are Associated with Increased Disease Severity in Corneal Ulcers.

The Steroids for Corneal Ulcers Trial (SCUT) was a multicenter, international study of bacterial keratitis in which 101 Pseudomonas aeruginosa infections were treated. Twenty-two of 101 P. aeruginosa isolates collected had a colony morphology characteristic of a loss-of-function mutation in lasR, the gene encoding a quorum-sensing master regulator. Ulcers caused by these 22 strains were associated with larger areas of corneal opacification, worse vision, and a lower rate of vision recovery in response to treatment than ulcers caused by the other isolates. The lasR sequences from these isolates each contained one of three nonsynonymous substitutions, and these strains were deficient in production of LasR-regulated protease and rhamnolipids. Replacement of lasR with either of the two most common lasR alleles from the SCUT isolates was sufficient to decrease protease and rhamnolipid production in PA14. Loss of LasR function is associated with increased production of CupA fimbriae, and the LasR-defective isolates exhibited higher production of CupA fimbriae than LasR-intact isolates. Strains with the same lasR mutation were of the same multilocus sequence type, suggesting that LasR-deficient, environmental P. aeruginosa strains were endemic to the area, and infections caused by these strains were associated with worse patient outcomes in the SCUT study. (This study has been registered at ClinicalTrials.gov under registration no. NCT00324168.) IMPORTANCE The LasR transcription factor is an important regulator of quorum sensing in P. aeruginosa and positively controls multiple virulence-associated pathways. The emergence of strains with lasR loss-of-function alleles in chronic disease is well described and is thought to represent a specific adaptation to the host environment. However, the prevalence and virulence of these strains in acute infections remain unclear. This report describes observations revealing that lasR mutants were common among isolates from a large, multicenter clinical study of keratitis and were associated with worse clinical outcomes than LasR-intact strains despite reduced production of LasR-regulated factors. Additionally, these lasR mutants were closely related strains or clones, as determined by molecular analysis. Because bacterial keratitis is community acquired, these data indicate infection by endemic, LasR-deficient strains in the environment. These results suggest that the conventional paradigm regarding the role for LasR-mediated regulation of virulence is more complex than previously appreciated.

Association of Biofilm Formation, Psl Exopolysaccharide Expression, and Clinical Outcomes in Pseudomonas aeruginosa Keratitis: Analysis of Isolates in the Steroids for Corneal Ulcers Trial.

IMPORTANCE: Bacterial virulence factors are increasingly recognized as important in the understanding of clinical infections. OBJECTIVE: To determine whether 2 potential virulence factors, in vitro biofilm formation and Psl exopolysaccharide (EPS) expression, influence clinical presentation or outcomes in Pseudomonas aeruginosa keratitis. DESIGN, SETTING, AND PARTICIPANTS: Laboratory investigation using P aeruginosa clinical isolates from the double-blind Steroids for Corneal Ulcers Trial (SCUT), which included patients at Aravind Eye Hospital, Proctor Foundation, University of California, San Francisco, and Dartmouth-Hitchcock Medical Center. SCUT was conducted from September 1, 2006, through February 22, 2010. All data used in this study were obtained during this period. Pseudomonas aeruginosa clinical isolates from SCUT were evaluated for in vitro biofilm formation, and Psl EPS expression was assessed using an anti-Psl monoclonal antibody (mAb) enzyme-linked immunosorbent assay. Planktonic growth kinetics and the susceptibility to anti-Psl mAb-mediated opsonophagocytic killing (OPK) were also evaluated in a subset of isolates. Linear regression assessed associations between SCUT patients' visual acuity and their corresponding biofilm formation and Psl EPS expression. Generalized estimating equation regression models were used to assess whether the change in visual acuity among SCUT patients was associated with Psl EPS expression or biofilm formation. MAIN OUTCOMES AND MEASURES: Biofilm formation, Psl production, OPK, and visual acuity. RESULTS: The P aeruginosa SCUT strains produced a mean (SD) in vitro biofilm score of 1.06 (0.32) (range 0.17-2.12). A 1-unit increase in biofilm was associated with a worse visual acuity of 2 lines measured in SCUT patients at baseline (0.20 logMAR; 95% CI, -0.03 to 0.44; P = .09) and 3 months (0.21 logMAR; 95% CI, 0.003 to 0.44; P = .047). Of 101 confirmed P aeruginosa SCUT isolates, 100 expressed Psl EPSs. In addition, all Psl-positive strains evaluated in the OPK assay were susceptible to anti-Psl mAb-mediated OPK. CONCLUSIONS AND RELEVANCE: The ability of P aeruginosa keratitis isolates to form biofilms in vitro was correlated with worse vision at presentation and after 3 months in SCUT. Ninety-nine percent of P aeruginosa keratitis isolates from SCUT produced Psl EPSs, and 100% of these evaluated Psl-positive isolates were susceptible to anti-Psl mAb-mediated OPK. These data indicate that biofilm formation and Psl EPSs may be candidate targets for novel therapeutics against P aeruginosa keratitis.

Tobramycin-Treated Pseudomonas aeruginosa PA14 Enhances Streptococcus constellatus 7155 Biofilm Formation in a Cystic Fibrosis Model System.

UNLABELLED: Cystic fibrosis (CF) is a human genetic disorder which results in a lung environment that is highly conducive to chronic microbial infection. Over the past decade, deep-sequencing studies have demonstrated that the CF lung can harbor a highly diverse polymicrobial community. We expanded our existing in vitro model of Pseudomonas aeruginosa biofilm formation on CF-derived airway cells to include this broader set of CF airway colonizers to investigate their contributions to CF lung disease, particularly as they relate to the antibiotic response of the population. Using this system, we identified an interspecies interaction between P. aeruginosa, a bacterium associated with declining lung function and worsening disease, and Streptococcus constellatus, a bacterium correlated with the onset of pulmonary exacerbations in CF patients. The growth rate and cytotoxicity of S. constellatus 7155 and P. aeruginosa PA14 were unchanged when grown together as mixed biofilms in the absence of antibiotics. However, the addition of tobramycin, the frontline maintenance therapy antibiotic for individuals with CF, to a mixed biofilm of S. constellatus 7155 and P. aeruginosa PA14 resulted in enhanced S. constellatus biofilm formation. Through a candidate genetic approach, we showed that P. aeruginosa rhamnolipids were reduced upon tobramycin exposure, allowing for S. constellatus 7155 biofilm enhancement, and monorhamnolipids were sufficient to reduce S. constellatus 7155 biofilm viability in the absence of tobramycin. While the findings presented here are specific to a biofilm of S. constellatus 7155 and P. aeruginosa PA14, they highlight the potential of polymicrobial interactions to impact antibiotic tolerance in unanticipated ways. IMPORTANCE: Deep-sequencing studies have demonstrated that the CF lung can harbor a diverse polymicrobial community. By recapitulating the polymicrobial communities observed in the CF lung and identifying mechanisms of interspecies interactions, we have the potential to select the best therapy for a given bacterial community and reveal potential opportunities for novel therapeutic interventions. Using an in vitro model of bacterial infection on CF airway cells, we tested how a particular polymicrobial community grows, damages human cells, and responds to antibiotics in single and mixed infections. We describe here the mechanism of an interspecies interaction between two pathogens in the CF lung, P. aeruginosa and S. constellatus, which is potentiated by a commonly prescribed antibiotic, tobramycin.

The Mitochondrial Genomes of the Nudibranch Mollusks, Melibe leonina and Tritonia diomedea, and Their Impact on Gastropod Phylogeny.

The phylogenetic relationships among certain groups of gastropods have remained unresolved in recent studies, especially in the diverse subclass Opisthobranchia, where nudibranchs have been poorly represented. Here we present the complete mitochondrial genomes of Melibe leonina and Tritonia diomedea (more recently named T. tetraquetra), two nudibranchs from the unrepresented Cladobranchia group, and report on the resulting phylogenetic analyses. Both genomes coded for the typical thirteen protein-coding genes, twenty-two transfer RNAs, and two ribosomal RNAs seen in other species. The twelve-nucleotide deletion previously reported for the cytochrome oxidase 1 gene in several other Melibe species was further clarified as three separate deletion events. These deletions were not present in any opisthobranchs examined in our study, including the newly sequenced M. leonina or T. diomedea, suggesting that these previously reported deletions may represent more recently divergent taxa. Analysis of the secondary structures for all twenty-two tRNAs of both M. leonina and T. diomedea indicated truncated d arms for the two serine tRNAs, as seen in some other heterobranchs. In addition, the serine 1 tRNA in T. diomedea contained an anticodon not yet reported in any other gastropod. For phylogenetic analysis, we used the thirteen protein-coding genes from the mitochondrial genomes of M. leonina, T. diomedea, and seventy-one other gastropods. Phylogenetic analyses were performed for both the class Gastropoda and the subclass Opisthobranchia. Both Bayesian and maximum likelihood analyses resulted in similar tree topologies. In the Opisthobranchia, the five orders represented in our study were monophyletic (Anaspidea, Cephalaspidea, Notaspidea, Nudibranchia, Sacoglossa). In Gastropoda, two of the three traditional subclasses, Opisthobranchia and Pulmonata, were not monophyletic. In contrast, four of the more recently named gastropod clades (Vetigastropoda, Neritimorpha, Caenogastropoda, and Heterobranchia) were all monophyletic, and thus appear to be better classifications for this diverse group.

Investigating the link between imipenem resistance and biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC ≤ 2 µg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC ≥ 8 µg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.

Circadian rhythms of crawling and swimming in the nudibranch mollusc Melibe leonina.

Daily rhythms of activity driven by circadian clocks are expressed by many organisms, including molluscs. We initiated this study, with the nudibranch Melibe leonina, with four goals in mind: (1) determine which behaviors are expressed with a daily rhythm; (2) investigate which of these rhythmic behaviors are controlled by a circadian clock; (3) determine if a circadian clock is associated with the eyes or optic ganglia of Melibe, as it is in several other gastropods; and (4) test the hypothesis that Melibe can use extraocular photoreceptors to synchronize its daily rhythms to natural light-dark cycles. To address these goals, we analyzed the behavior of 55 animals exposed to either artificial or natural light-dark cycles, followed by constant darkness. We also repeated this experiment using 10 animals that had their eyes removed. Individuals did not express daily rhythms of feeding, but they swam and crawled more at night. This pattern of locomotion persisted in constant darkness, indicating the presence of a circadian clock. Eyeless animals also expressed a daily rhythm of locomotion, with more locomotion at night. The fact that eyeless animals synchronized their locomotion to the light-dark cycle suggests that they can detect light using extraocular photoreceptors. However, in constant darkness, these rhythms deteriorated, suggesting that the clock neurons that influence locomotion may be located in, or near, the eyes. Thus, locomotion in Melibe appears to be influenced by both ocular and extraocular photoreceptors, although the former appear to have a greater influence on the expression of circadian rhythms.