Coaggregation is the specific recognition and adherence of genetically distinct microorganisms. Because most biofilms are polymicrobial communities, there is potential for coaggregation to play an integral role in spatiotemporal biofilm development and the moderation of biofilm community composition. However, understanding of the mechanisms contributing to coaggregation and the relevance of coaggregation to biofilm ecology is at a very early stage. The purpose of this review is to highlight recent advances in the understanding of microbial coaggregation within different environments and to describe the possible ecological ramifications of such interactions. Bacteria that coaggregate with many partner species within different environments will be highlighted, including oral streptococci and oral bridging organisms such as fusobacteria, as well as the freshwater sphingomonads and acinetobacters. Irrespective of environment, it is proposed that coaggregation is essential for the orchestrated development of multi-species biofilms.

Resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) may be an important contributor to the ability of Listeria spp. to persist in the processing plant environment. Although a plasmid-borne disinfectant resistance cassette (bcrABC) has been identified in Listeria monocytogenes, horizontal transfer of these genes has not been characterized. Nonpathogenic Listeria spp. such as L. innocua and L. welshimeri are more common than L. monocytogenes in food processing environments and may contribute to the dissemination of disinfectant resistance genes in listeriae, including L. monocytogenes. In this study, we investigated conjugative transfer of resistance to BC and to cadmium from nonpathogenic Listeria spp. to other nonpathogenic listeriae, as well as to L. monocytogenes. BC-resistant L. welshimeri and L. innocua harboring bcrABC, along with the cadmium resistance determinant cadA2, were able to transfer resistance to other nonpathogenic listeriae as well as to L. monocytogenes of diverse serotypes, including strains from the 2011 cantaloupe outbreak. Transfer among nonpathogenic Listeria spp. was noticeably higher at 25°C than at 37°C, whereas acquisition of resistance by L. monocytogenes was equally efficient at 25 and 37°C. When the nonpathogenic donors were resistant to both BC and cadmium, acquisition of cadmium resistance was an effective surrogate for transfer of resistance to BC, suggesting coselection between these resistance attributes. The results suggest that nonpathogenic Listeria spp. may behave as reservoirs for disinfectant and heavy metal resistance genes for other listeriae, including the pathogenic species L. monocytogenes.

BACKGROUND Bacillus anthracis, the causative agent of anthrax, is a potential bioterrorism agent. Anthrax meningitis may be a manifestation of B. anthracis infection, has high mortality, and requires more aggressive treatment than anthrax without meningitis. Rapid identification and treatment of anthrax meningitis are essential for successful management of an anthrax mass casualty incident. METHODS Three hundred six published reports from 1880 through 2013 met pre-defined 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 are proposed as a four-item screening tool for use during mass casualty incidents. Presence of any one 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 ([LR−]=0.12 [0.19] for adult [pediatric] cohorts), while presence of two or more factors made meningitis very likely ([LR+]=26.5 [29.2]). Survival of anthrax meningitis was predicted by treatment with a bactericidal agent (P=0.005) and use of multiple antimicrobials (P=0.012). CONCLUSIONS We developed an evidence-based triage 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.

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.

While the early stages of biofilm formation have been well characterized, less is known about the requirements for Pseudomonas aeruginosa to maintain a mature biofilm. We utilized a P. aeruginosa-phage interaction to identify rmcA and morA, two genes which encode bis-(3′,5′)-cyclic dimeric GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and are important for the regulation of biofilm maintenance. Deletion of these genes initially results in an elevated biofilm phenotype characterized by increased production of c-di-GMP, Pel polysaccharide, and/or biofilm biomass. In contrast to the wild-type strain, these mutants were unable to maintain the biofilm when exposed to carbon-limited conditions. The susceptibility to nutrient limitation, as well as subsequent loss of biofilm viability of these mutants, was phenotypically reproduced with a stringent response mutant (ΔrelA ΔspoT), indicating that the ΔrmcA and ΔmorA mutants may be unable to appropriately respond to nutrient limitation. Genetic and biochemical data indicate that RmcA and MorA physically interact with the Pel biosynthesis machinery, supporting a model whereby unregulated Pel biosynthesis contributes to the death of the ΔrmcA and ΔmorA mutant strains in an established biofilm under nutrient limitation. These findings provide evidence that c-di-GMP-mediated regulation is required for mature biofilms of P. aeruginosa to effectively respond to changing availability of nutrients. Furthermore, the PDEs involved in biofilm maintenance are distinct from those required for establishing a biofilm, suggesting that a wide variety of c-di-GMP metabolizing enzymes in organisms such as P. aeruginosa allows for discrete control over the formation, maintenance or dispersion of biofilms. IMPORTANCE Recent advances in our understanding of c-di-GMP signaling have provided key insights into the regulation of biofilms. Despite an improved understanding of how biofilms initially form, the processes that facilitate the long-term maintenance of these multicellular communities remain opaque. We found that P. aeruginosa requires two phosphodiesterases, RmcA and MorA, to maintain a mature biofilm and that biofilms lacking these PDEs succumb to nutrient limitation and die. The biofilm maintenance deficiency observed in ΔrmcA and ΔmorA mutants was also found in the stringent response-defective ΔrelA ΔspoT strain, suggesting that a regulatory intersection between c-di-GMP signaling, extracellular polysaccharide biosynthesis, and the nutrient limitation response is important for biofilm persistence. We uncover components of an important regulatory system needed for P. aeruginosa biofilms to persist in nutrient-poor conditions and provide some of the first evidence that maintaining a mature biofilm is an active process.

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.

Background 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 two 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. Methods 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. Results A total of 149 cases of systemic anthrax were identified (cutaneous [n=59], gastrointestinal [n=28], inhalation [n=26], primary anthrax meningitis [n=19], multiple routes [n=9], and injection [n=8]). 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 a total of ≥3 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. Conclusion Combination bactericidal and protein synthesis inhibitor therapy may be appropriate in severe anthrax disease, particularly anthrax meningitis, in a mass casualty incident.

Pseudomonas aeruginosa forms surface-attached communities that persist in the face of antimicrobial agents and environmental perturbation. Published work has found extracellular polysaccharide (EPS) production, regulation of motility and induction of stress response pathways as contributing 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 Δ yfiN mutant exhibit a maintenance defect, which can be further exacerbated by exposure to peroxide (H 2 O 2 ); this defect can be rescued through genetic complementation. Additionally, we found that the Δ yfiN mutant biofilms produce less c-di-GMP than WT, and that H 2 O 2 treatment enhanced motility of surface-associated bacteria and increased cell death for the Δ yfiN 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 P. 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 to insure 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.