Immunization with a Biofilm-Disrupting Nontypeable Haemophilus influenzae Vaccine Antigen Did Not Alter the Gut Microbiome in Chinchillas, Unlike Oral Delivery of a Broad-Spectrum Antibiotic Commonly Used for Otitis Media

Abstract

The use of broad-spectrum antibiotics to treat diseases, such as the highly prevalent pediatric disease otitis media (OM), contributes significantly to the worldwide emergence of multiple-antibiotic-resistant microbes, and gut dysbiosis with diarrhea is a common adverse sequela. Moreover, for many diseases, like OM, biofilms contribute significantly to chronicity and recurrence, yet biofilm-resident bacteria are characteristically highly resistant to antibiotics. The most cost-effective way to both prevent and resolve diseases like OM, as well as begin to address the problem of growing antibiotic resistance, would be via the development of novel approaches to eradicate bacterial biofilms. Toward this goal, we designed a vaccine antigen that induces the formation of antibodies that prevent biofilm formation and, thereby, experimental OM in the middle ears of chinchillas by the predominant Gram-negative pathogen responsible for this disease, nontypeable Haemophilus influenzae. These antibodies also significantly disrupt preexisting biofilms formed by diverse pathogens. Whereas preclinical data strongly support the continued development of this vaccine antigen, which targets an essential structural element of bacterial biofilms, a concern has been whether active immunization would also lead to unintended collateral damage in the form of an altered gut microbiome. To address this concern, we assessed changes in the microbiome of the chinchilla gut over time after the delivery of either amoxicillin-clavulanate, the standard of care for OM, or after immunization with our biofilm-targeted vaccine antigen either via a traditional subcutaneous route or via a novel noninvasive transcutaneous route. We show that differences in the abundance of specific taxa were found only in the stools of antibiotic-treated animals. IMPORTANCE The prevalence of chronic and recurrent diseases, combined with the overuse/abuse of antibiotics that has led to the sobering emergence of bacteria resistant to multiple antibiotics, has mandated that we develop novel approaches to better manage these diseases or, ideally, prevent them. Biofilms play a key role in the pathogenesis of chronic and recurrent bacterial diseases but are difficult, if not impossible, to eradicate with antibiotics. We developed a vaccine antigen designed to mediate biofilm disruption; however, it is also important that delivery of this vaccine does not induce collateral damage to the microbiome. The studies described here validated a vaccine approach that targets biofilms without the consequences of an altered gut microbiome. While delivery of the antibiotic most commonly given to children with ear infections did indeed alter the gut microbiome, as expected, immunization via traditional injection or by noninvasive delivery to the skin did not result in changes to the chinchilla gut microbiome.

Bacterial Biofilms Utilize an Underlying Extracellular DNA Matrix Structure That Can Be Targeted for Biofilm Resolution

“…To address concerns about the potential to induce dysbiosis of the gut flora following active immunization with a vaccine antigen directed against a biofilm constituent (given that there are many 'good' biofilms on and in the human body as well), we immunized cohorts of chinchillas both via an subcutaneous and transcutaneous route with the same tip-chimer peptide to compare their gut microbiome to those that had been treated with the standard-of-care for OME (e.g., amoxicllin/clavulanic acid). We found that whereas orally administered amoxicllin/clavulanic did indeed rapidly induce dysbiosis of the gut flora, as expected, neither route of immunization with the DNABII-directed immunogen did so [56] which suggested that the induced antibodies did not likely transude onto the non-inflamed (non-diseased) mucosa that lined the GI tract with beneficial biofilms in these animals.…”

Nontypeable Haemophilus influenzae newly released (NRel) from biofilms by antibody-mediated dispersal versus antibody-mediated disruption are phenotypically distinct

“…Whereas their focus was on antibodies that reduce carriage, and thus transmission of antibiotic-resistant bacteria [ 103 ], we envision use of specifically induced antibodies to release biofilm-resident NTHI from these highly resistant communities so they can be killed by host immune effectors and when necessary, traditional antibiotics, with the latter now used at a markedly reduced dose and for a shorter course due to the highly sensitive phenotype of NRel NTHI. An additional potential benefit of a less frequent antibiotic treatment regimen is reduction of off-target side effects and other undesirable sequelae of oral antibiotic use; which includes development of antibiotic resistance [ 84 , 104 ] and/or disruption of the gut microbiome [ 16 , [105] , [106] , [107] , [108] ].…”

Short Term Effect of Ivermectin on the Bacterial Microbiota from Fecal Samples in Chinchillas (Chinchilla lanigera)

“…Chinchillas were placed individually into clean cages, and fecal pellets were collected upon evacuation. Following collection, these samples were transferred immediately into sterile cryogenic vials by sterile forceps and stored at −80 °C until microbiota analysis [ 14 ].…”