Architecture of cell-cell junctions in situ reveals a mechanism for bacterial biofilm inhibition.

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam-milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.

Biofilm Maintenance as an Active Process: Evidence that Biofilms Work Hard to Stay Put.

Biofilm formation represents a critical strategy whereby bacteria can tolerate otherwise damaging environmental stressors and antimicrobial insults. While the mechanisms bacteria use to establish a biofilm and disperse from these communities have been well-studied, we have only a limited understanding of the mechanisms required to maintain these multicellular communities. Indeed, until relatively recently, it was not clear that maintaining a mature biofilm could be considered an active, regulated process with dedicated machinery. Using Pseudomonas aeruginosa as a model system, we review evidence from recent studies that support the model that maintenance of these persistent, surface-attached communities is indeed an active process. Biofilm maintenance mechanisms include transcriptional regulation and second messenger signaling (including the production of extracellular polymeric substances). We also discuss energy-conserving pathways that play a key role in the maintenance of these communities. We hope to highlight the need for further investigation to uncover novel biofilm maintenance pathways and suggest the possibility that such pathways can serve as novel antibiofilm targets.

The Diguanylate Cyclase YfiN of Pseudomonas aeruginosa Regulates Biofilm Maintenance in Response to Peroxide.

Pseudomonas aeruginosa forms surface-attached communities that persist in the face of antimicrobial agents and environmental perturbation. Published work has found that extracellular polysaccharide (EPS) production, regulation of motility, and induction of stress response pathways contribute to biofilm tolerance during such insults. However, little is known regarding the mechanism(s) whereby biofilm maintenance is regulated when exposed to such environmental challenges. Here, we provide evidence that the diguanylate cyclase YfiN is important for the regulation of biofilm maintenance when exposed to peroxide. We find that compared to the wild type (WT), static biofilms of the ΔyfiN mutant exhibit a maintenance defect, which can be further exacerbated by exposure to peroxide (H2O2); this defect can be rescued through genetic complementation. Additionally, we found that the ΔyfiN mutant biofilms produce less c-di-GMP than WT and that H2O2 treatment enhanced motility of surface-associated bacteria and increased cell death for the ΔyfiN mutant grown as a biofilm compared to WT biofilms. These data provide evidence that YfiN is required for biofilm maintenance by P. aeruginosa, via c-di-GMP signaling, to limit motility and protect viability in response to peroxide stress. These findings add to the growing recognition that biofilm maintenance by P. aeruginosa is an actively regulated process that is controlled, at least in part, by the wide array of c-di-GMP metabolizing enzymes found in this microbe. IMPORTANCE We build on previous findings that suggest that Pseudomonas aeruginosa utilizes c-di-GMP metabolizing enzymes to actively maintain a mature biofilm. Here, we explore how the diguanylate cyclase YfiN contributes to the regulation of biofilm maintenance during peroxide exposure. We find that mature P. aeruginosa biofilms require YfiN to synthesize c-di-GMP, regulate motility, and ensure viability during peroxide stress. These findings provide further evidence that the modulation of c-di-GMP in response to environmental signals is an important mechanism by which biofilms are maintained.

Differential Surface Competition and Biofilm Invasion Strategies of Pseudomonas aeruginosa PA14 and PAO1.

Pseudomonas aeruginosa strains PA14 and PAO1 are among the two best-characterized model organisms used to study the mechanisms of biofilm formation while also representing two distinct lineages of P. aeruginosa. Previous work has shown that PA14 and PAO1 use different strategies for surface colonization; they also have different extracellular matrix composition and different propensities to disperse from biofilms back into the planktonic phase surrounding them. We expand on this work here by exploring the consequences of these different biofilm production strategies during direct competition. Using differentially labeled strains and microfluidic culture methods, we show that PAO1 can outcompete PA14 in direct competition during early colonization and subsequent biofilm growth, that they can do so in constant and perturbed environments, and that this advantage is specific to biofilm growth and requires production of the Psl polysaccharide. In contrast, P. aeruginosa PA14 is better able to invade preformed biofilms and is more inclined to remain surface-associated under starvation conditions. These data together suggest that while P. aeruginosa PAO1 and PA14 are both able to effectively colonize surfaces, they do so in different ways that are advantageous under different environmental settings. IMPORTANCE Recent studies indicate that P. aeruginosa PAO1 and PA14 use distinct strategies to initiate biofilm formation. We investigated whether their respective colonization and matrix secretion strategies impact their ability to compete under different biofilm-forming regimes. Our work shows that these different strategies do indeed impact how these strains fair in direct competition: PAO1 dominates during colonization of a naive surface, while PA14 is more effective in colonizing a preformed biofilm. These data suggest that even for very similar microbes there can be distinct strategies to successfully colonize and persist on surfaces during the biofilm life cycle.

Identifying Meningitis During an Anthrax Mass Casualty Incident: Systematic Review of Systemic Anthrax Since 1880.

BACKGROUND: Bacillus anthracis, the causative agent of anthrax, is a potential bioterrorism agent. Anthrax meningitis is a common manifestation of B. anthracis infection, has high mortality, and requires more aggressive treatment than anthrax without meningitis. Its rapid identification and treatment are essential for successful management of an anthrax mass casualty incident. METHODS: Three hundred six published reports from 1880 through 2013 met predefined inclusion criteria. We calculated descriptive statistics for abstracted cases and conducted multivariable regression on separate derivation and validation cohorts to identify clinical diagnostic and prognostic factors for anthrax meningitis. RESULTS: One hundred thirty-two of 363 (36%) cases with systemic anthrax met anthrax meningitis criteria. Severe headache, altered mental status, meningeal signs, and other neurological signs at presentation independently predicted meningitis in the derivation cohort and were tested as a 4-item assessment tool for use during anthrax mass casualty incidents. Presence of any 1 factor on admission had a sensitivity for finding anthrax meningitis of 89% (83%) in the adult (pediatric) validation cohorts. Anthrax meningitis was unlikely in the absence of any of these signs or symptoms (likelihood ratio [LR]- = 0.12 [0.19] for adult [pediatric] cohorts), while presence of 2 or more made meningitis very likely (LR+ = 26.5 [30.0]). Survival of anthrax meningitis was predicted by treatment with a bactericidal agent (P = .005) and use of multiple antimicrobials (P = .01). CONCLUSIONS: We developed an evidence-based assessment tool for screening patients for meningitis during an anthrax mass casualty incident. Its use could improve both patient outcomes and resource allocation in such an event.

Postexposure Prophylaxis After Possible Anthrax Exposure: Adherence and Adverse Events.

Anthrax postexposure prophylaxis (PEP) was recommended to 42 people after a laboratory incident that involved potential aerosolization of Bacillus anthracis spores in 2 laboratories at the Centers for Disease Control and Prevention in 2014. At least 31 (74%) individuals who initiated PEP did not complete either the recommended 60 days of antimicrobial therapy or the 3-dose vaccine regimen. Among the 29 that discontinued the antimicrobial component of PEP, most (38%) individuals discontinued PEP because of their low perceived risk of infection; 9 (31%) individuals discontinued prophylaxis due to PEP-related minor adverse events, and 10% cited both low risk and adverse events as their reason for discontinuation. Most minor adverse events reported were gastrointestinal complaints, and none required medical attention. Individuals taking ciprofloxacin were twice as likely (RR = 2.02, 95% CI = 1.1-3.6) to discontinue antimicrobial prophylaxis when compared to those taking doxycycline. In the event anthrax PEP is recommended, public health messages and patient education materials will need to address potential misconceptions regarding exposure risk and provide information about possible adverse events in order to promote PEP adherence.

Antimicrobial Treatment for Systemic Anthrax: Analysis of Cases from 1945 to 2014 Identified Through a Systematic Literature Review.

Systemic anthrax is associated with high mortality. Current national guidelines, developed for the individualized treatment of systemic anthrax, outline the use of combination intravenous antimicrobials for a minimum of 2 weeks, bactericidal and protein synthesis inhibitor antimicrobials for all cases of systemic anthrax, and at least 3 antimicrobials with good blood-brain barrier penetration for anthrax meningitis. However, in an anthrax mass casualty incident, large numbers of anthrax cases may create challenges in meeting antimicrobial needs. To further inform our understanding of the role of antimicrobials in treating systemic anthrax, a systematic review of the English-language literature was conducted to identify cases of systemic anthrax treated with antimicrobials for which a clinical outcome was recorded. A total of 149 cases of systemic anthrax were identified. Among the identified 59 cases of cutaneous anthrax, 33 were complicated by meningitis (76% mortality), while 26 simply had evidence of the systemic inflammatory response syndrome (4% mortality); 21 of 26 (81%) of this latter group received monotherapy. Subsequent analysis regarding combination antimicrobial therapy was restricted to the remaining 123 cases of more severe anthrax (overall 67% mortality). Recipients of combination bactericidal and protein synthesis inhibitor therapy had a 45% survival versus 28% in the absence of combination therapy (p = 0.07). For meningitis cases (n = 77), survival was greater for those receiving 3 or more antimicrobials over the course of treatment (3 of 4; 75%), compared to receipt of 1 or 2 antimicrobials (12 of 73; 16%) (p = 0.02). Median parenteral antimicrobial duration was 14 days. Combination bactericidal and protein synthesis inhibitor therapy may be appropriate in severe anthrax disease, particularly anthrax meningitis, in a mass casualty incident.