Keywords::
It is now clear that biofilm communities are an important determinant of bacterial persistence during chronic and/or recurrent infections Citation[1]. Epidemiological data indicate that many chronic infections such as otitis media (OM) are polymicrobial Citation[2], and it has long been appreciated that virulence and/or resistance to treatment can be dramatically impacted by coinfection Citation[3]. Our recent work shows that, in the context of experimental OM, coinfection promotes bacterial persistence and antibiotic resistance via interspecies communication (i.e., quorum signaling) Citation[4]. Thus, targeting bacterial communication is a potential means for primary antimicrobial treatment or as an adjunct to antibiotic therapy.
Otitis media
Otitis media is an extremely common pediatric ailment Citation[5], caused mostly by Haemophilus influenzae, Streptococcus pneumoniae and/or Moraxella catarrhalis. Most chronic OM infections are polymicrobial Citation[2], and combinations of H. influenzae, M. catarrhalis and S. pneumoniae have been detected in patient samples from chronic and recurrent OM Citation[2,6]. It is important to consider the possibility that polymicrobial infection impacts severity of disease and persistence of otopathogens. It is also well-documented that polymicrobial infection impacts treatment efficacy Citation[3,4,7–11], thus becoming an important consideration for development of new therapeutics.
Otitis media presentations vary from acute to chronic infections, and can be highly resistant to both immune clearance and antibiotic treatment Citation[12–14]. Chronic and recurrent OM involve persistence of bacteria within biofilm communities Citation[15–17]. Our recent work shows that polymicrobial biofilms impact bacterial persistence and antibiotic resistance Citation[4,18]. Thus, insight into the mechanisms of biofilm formation during single species and polymicrobial infection may provide new targets for treatment of OM.
Conventional treatment of otitis media
Treatment of OM varies depending on symptoms, severity and recurrence of the infection. Acute episodes and OM with effusion are often managed through watchful waiting, prescription of antibiotics and/or use of oral steroids. Chronic OM may warrant further interventions such as myringotomy for ventilation, placement of tympanostomy tubes and/or adenoidectomy.
Antibiotic treatment of OM is extremely common Citation[19], with β-lactams being the most frequently prescribed drugs Citation[20]. Approximately 13% of all acute OM episodes result in treatment failure or recurrence, regardless of the treatment method Citation[20], and the failure rate for bacteriological eradication is strikingly high for pneumococci and H. influenzaeCitation[21,22]. Other studies have shown that antibiotic treatment provides only minimal and transient benefit to patients experiencing OM with effusion Citation[23]. Biofilm formation by otopathogens and polymicrobial infection also contribute to antibiotic treatment failure via secretion of β-lactamase or protection of susceptible organisms within the biofilm Citation[4,24].
Owing to the high rate of treatment failure and the selective pressure exerted by antibiotic treatment, attention has been focused on vaccination to prevent OM and therefore reduce the need for antibiotics Citation[25]. Despite the reduction in disease caused by certain pneumococcal serotypes, replacement of vaccine serotypes with nonvaccine serotypes and increased incidence of OM caused by other otopathogens have complicated the impact of these vaccines on carriage and chronic infection Citation[26]. There clearly remains a pressing need for alternative and effective OM treatments that exert minimal selective pressure towards resistance.
Targeting bacterial communication
Biofilm formation by many bacterial species is controlled in part through cell density-dependent quorum signaling networks Citation[27–30]. Signaling networks include acyl homoserine lactones (AHL) in Gram-negative bacteria, species-specific peptide autoinducers in Gram-positive bacteria, and autoinducer-2 (AI-2) in both Gram-positive and Gram-negative bacteria (see Citation[31] for review). AHL signaling as well as peptide autoinducers generally mediate intraspecies communication. AI-2, however, is a ribose-derived signaling mediator that is commonly referred to as an interspecies signal Citation[28,30]. Recent efforts to disrupt bacterial communication have mostly focused on inhibiting AHL and peptide autoinducer signaling Citation[32,33], but the interspecies aspect of AI-2 makes this molecule an attractive target for treating polymicrobial infection.
AI-2 influences biofilm development for many species, and in some instances can impact development of polymicrobial biofilms Citation[34,35]. AI-2 quorum signaling influences biofilm formation and disease severity of H. influenzaeCitation[36,37] and M. catarrhalisCitation[4], even though the latter species does not possess the genetic determinant for AI-2 production (luxS). S. pneumoniae produces AI-2, and mutation of luxS reduces virulence and persistence in a murine model of nasopharyngeal carriage Citation[38,39]. In polymicrobial OM, we showed that AI-2 produced by H. influenzae promoted M. catarrhalis biofilm formation, antibiotic resistance and persistence in the chinchilla model of OM Citation[4]. These data show that H. influenzae and M. catarrhalis utilize AI-2 to establish biofilms and persistent infections. Thus, disruption of AI-2 signaling may also disrupt biofilm formation and infection by these pathogens. Targeting bacterial communication through AI-2 quorum signaling may therefore represent a significant advance in treatment of chronic or recurrent OM infections, particularly those of a polymicrobial nature.
As all three of the leading OM pathogens appear to utilize the AI-2 signal, one target for blocking bacterial communication would be interfering with recognition of AI-2. In Vibrio harveyi, sensing of AI-2 occurs through a two-component system involving LuxP and LuxQ Citation[40], where LuxP binds AI-2 and LuxQ senses and propagates the signal through its sensor kinase and response regulator domains Citation[41]. Outside of Vibrio, many species possess the Lsr ABC transporter for binding of AI-2 by LsrB and transport through a heterodimeric membrane channel Citation[42–45]. In addition to LsrB, the ribose binding protein RbsB also binds AI-2 Citation[46,47]. Thus, ribose transport systems may also participate in the internalization of, or response to, AI-2 and would therefore represent additional targets for disruption of AI-2 quorum signaling.
Many of the current AI-2 nucleoside analogues aimed at disrupting AI-2 transport, such as adenosine derivatives, only antagonize the V. harveyi LuxPQ sensor system Citation[48]. Other AI-2 analogs have been shown to antagonize signaling in Salmonella typhimurium and are nontoxic toward mammalian cells, but activated signaling in V. harveyi in the presence of the AI-2 precursor 4,5-dihydroxy-2,3-pentanedion (DPD) Citation[49]. Another complication in targeting AI-2 transport or sensor systems is the duration of inhibition. For alkyl-DPDs, inhibition of V. harveyi signaling lasted less than 2 h Citation[50], limiting their therapeutic potential.
Another target for disrupting bacterial communication would be inhibition of AI-2 production. This strategy may also disrupt bacterial metabolism as LuxS plays an intricate role in the activated methyl cycle (see Citation[51] for review). Research focusing on the mechanism of action of LuxS has identified key amino acid residues and catalytic intermediates that could be suitable targets for inhibiting LuxS Citation[52]. LuxS substrate analogues of S-ribosyl homocysteine (SRH) acted as time-dependent inhibitors of AI-2 production in V. harveyi, Bacillus subtilis and Escherichia coli. It may therefore be possible to generate modified SRH analogues that permanently inhibit LuxS and AI-2 production.
Another strategy to block bacterial communication would be external quenching of AI-2. This could be accomplished through degradation or cleavage of AI-2, use of AI-2 binding proteins, or alteration of AI-2 to limit sensing of the signal. One example of AI-2 quenching in the literature is the use of E. coli AI-2 kinase (LsrK) for in vitro phosphorylation of AI-2 Citation[53]. According to this study, the addition of LsrK and ATP to bacterial cultures generated phospho-AI-2 that could not be transported into bacterial cells, and degraded over time without stimulating quorum signaling pathways. Such strategies may prove the most effective against otopathogens as LsrK can phosphorylate DPD, the precursor of all discrete structures of AI-2, thus targeting bacterial communication regardless of the specific AI-2 structure or transport/sensor mechanisms present during the infection.
Challenges
Several challenges exist in the development of effective OM treatments targeting bacterial communication, including obstacles in specificity and delivery of the treatment, potential negative effects of AI-2 inhibition in some otopathogens, and the implications of systemic inhibition of AI-2 quorum signaling. Therapy aimed at blocking AI-2 transport systems would need to account for differences in the discrete structure of AI-2 utilized by each otopathogen. All therapeutics targeting AI-2 signaling would ideally penetrate the biofilms that form during chronic infection, and LuxS inhibitor compounds would also need to be transported into the bacterial cell to be effective.
Due to the interspecies nature of AI-2 signaling, targeting AI-2 has implications beyond the common otopathogens. Disruption of AI-2 can promote virulence or virulence-related phenotypes for Staphylococcus aureus and Streptococcus pyogenesCitation[54,55], species found within the upper airways that occasionally cause OM. Another consideration for interference with AI-2 signaling concerns the potential impact on the normal microbiota. AI-2 is produced by avirulent species within the oral and gastrointestinal microbiota Citation[56,57]. There is limited information concerning AI-2 quorum signaling in these populations, but the possibility exists that AI-2 signaling may be critical for the establishment or maintenance of normal microbiota and defense against pathogens. Thus, it would be ideal to engineer an AI-2 inhibitor that remains localized to the site of infection.
Despite the challenges in manipulating bacterial communication, AI-2 remains an intriguing target for disruption of chronic, recurrent and polymicrobial infections. Limiting bacterial communication may be sufficient to reduce or eliminate key species and make biofilms more susceptible to immune clearance or antibiotics. Thus, disruption of polymicrobial biofilms in this manner represents a potential means to reduce antibiotic treatment failure.
Financial & competing interests disclosure
The work in W Edward Swords’ laboratory is funded by research grants from NIH/NIDCD (DC007444 and DC10051). Chelsie Armbruster is supported by an NIH training grant (T32 AI07401, Steven Mizel, Principal Investigator). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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