Bacterial Biota of Women with Bacterial Vaginosis Treated with Lactoferrin: an Open Prospective Randomized Trial Alessandra Pinoa, Giuliana Giuntab, Cinzia L
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MICROBIAL ECOLOGY IN HEALTH AND DISEASE, 2017 VOL. 28, 1357417 https://doi.org/10.1080/16512235.2017.1357417 RESEARCH ARTICLE Bacterial biota of women with bacterial vaginosis treated with lactoferrin: an open prospective randomized trial Alessandra Pinoa, Giuliana Giuntab, Cinzia L. Randazzoa, Salvatore Caruso b, Cinzia Caggia a and Antonio Ciancib aDepartment of Agriculture, Food and Environment (Di3A), University of Catania, Catania, Italy; bDepartment of General Surgery and Medical Surgical Specialties, Gynecological Clinic, University of Catania, Policlinico Universitario, Catania, Italy ABSTRACT ARTICLE HISTORY Background: Bacterial vaginosis is the most frequent condition associated to the vaginal Received 2 March 2017 microbiota imbalance, affecting about the 40–50% of women in the world. Even if antibiotics Accepted 14 July 2017 are effcetive for bacterial vaginosis treatment a long-term recurrence rates, higher than 70%, KEYWORDS is recorded. Lactoferrin is an iron-binding glycoprotein with bacteriostatic and bactericidal Vaginal microbiota; Ion properties. It owns the ability to protect the host against infection, by binding and regulating Torrent; 16S rDNA; vaginal the iron needed for the bacterial proliferation. infection; lactoferrin; in vivo Objective: The present study was an open prospective randomized trial (registration no. SHI- trial; Lactobacillus spp.; EVE-2014.01) aimed at characterizing the bacterial biota of women affected by bacterial L. helveticus vaginosis (BV) and assessing the effects of two different lactoferrin concentrations (100 mg and 200 mg vaginal pessaries) on the composition and dynamics of the vaginal bacterial biota. Design: Sixty women with BV were recruited and randomized into two groups to receive lactoferrin pessaries for 10 days. Clinical evaluation was based on Amsel criteria and Nugent scores. Culture-dependent methods and Ion Torrent PGM sequencing of the 16S rRNA gene were applied to study in depth the overall structure of the vaginal bacterial biota and its dynamics during the treatment. Results: Vaginal lactoferrin administration modified the vaginal microbiota composition in patients with BV. During treatment, both 100 mg and 200 mg lactoferrin vaginal pessaries significantly decreased the occurrence of bacteria associated with BV, such as Gardnerella, Prevotella, and Lachnospira, and increased the occurrence of Lactobacillus species. The bac- terial biota balance was maintained up to 2 weeks after treatment only in women treated with 200 mg lactoferrin pessaries. Conclusions: This study indicates that lactoferrin could be proposed as an alternative therapeutic approach for BV. Our data showed, for the first time, the dominance of Lactobacillus helveticus species during and after vaginal lactoferrin treatment. Introduction play a protective role against pathogens by different mechanisms such as the production of lactic acid, The human vaginal microbiota is a complex ecosys- resulting in a low pH (3.5–4.5) [5,6]; the enhance- tem that plays an important role in women’s health, ment on the host’s innate immune system [7]; and having a balanced mutualistic association with the the production of antimicrobial compounds, includ- vaginal environment. In this relationship, the host ing target-specific bacteriocins [8,9] and the broad- provides benefit to the microbial communities in spectrum hydrogen peroxide (H O )[6,10]. The most the form of nutrients needed to support bacterial 2 2 frequent condition associated with imbalance in the growth, and the vaginal microbiota plays a protective vaginal microbiota is bacterial vaginosis (BV), which role in preventing colonization by potentially patho- affects about 40–50% of women in the world [11–13]. genic organisms [1]. Several studies have shown that The etiology of BV is unclear, but it is currently a healthy vaginal environment is dominated by lacto- considered a polymicrobial disorder in which lacto- bacilli [2], mainly Lactobacillus crispatus, L. gasseri, L. bacilli are reduced, absent, or lacking specific antimi- jensenii, and L. iners, followed by L. acidophilus, L. crobial properties (i.e. production of H O ) with a fermentum, L. plantarum, L. brevis, L. casei, L. vagi- 2 2 concurrent increase in opportunistic pathogenic bac- nalis, L. delbrueckii, L. salivarius, L. reuteri, and L. teria, mainly Gardnerella vaginalis but also anaerobes rhamnosus species [3,4]. These microorganisms, such as Atopobium vaginae, Bacteroides, Mobiluncus, which represent 80–95% of the resident bacteria, Prevotella, Peptostreptococcus spp., Ureaplasma CONTACT Cinzia L. Randazzo [email protected] Department of Agriculture, Food and Environment (Di3A), University of Catania Santa Sofia street 98, 95123 Catania, Italy Supplemental material for this article can be accessed here. © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 A. PINO ET AL. urealyticum, and Mycoplasma hominis [1,14,15]. 200 mg) on the vaginal bacterial biota. For this pur- Treatment with antibiotics, such as metronidazole pose, the vaginal microbiota composition and and clindamycin, is indicated in both symptomatic dynamics of BV-positive women were investigated and asymptomatic women affected by BV [15,16]. before, during, and after lactoferrin administration, Even if antibiotic medication is effective in treating using an integrated approach based on culture-depen- BV, about 25% of women will develop a new episode dent methods and Ion Torrent™ PGM™ sequencing of of BV within 4 weeks [17,18] and the long-term 16S ribosomal RNA (rRNA) gene-based amplicons. recurrence rates are > 70% [15,19]. Moreover, there are several disadvantages associated with antibiotic therapies, including superinfections with pathogenic Materials and methods microorganisms, susceptibility of lactobacilli to clin- Study design and patient enrollment damycin, and an increased drug resistance by vaginal pathogens, particularly G. vaginalis and anaerobic The present study was an open prospective rando- bacteria [15,20–22]. For these reasons, alternative mized trial with two parallel groups. From October therapeutic agents need to be sought. 2015 to May 2016, 60 BV-positive women were Lactoferrin is an approximately 80 kDa iron-binding recruited at the Department of General Surgery glycoprotein belonging to the transferrin family, which and Medical Surgical Specialties, Gynecological is produced and stored in specific (secondary) neutro- Clinic, University of Catania (Catania, Italy). phil granules and released during neutrophil activation Participants were randomly (1:1 ratio) allocated to and degranulation. Lactoferrin has bacteriostatic and one of the two experimental groups, A or B, and bactericidal properties, with the ability to protect the treated for 10 days with 200 mg or 100 mg lacto- host against infection, by binding and regulating the ferrin, respectively. Vaginal lactoferrin pessaries iron needed for bacterial proliferation [23–25]. The were kindly provided by AG Pharma s.r.l. (Rome, antimicrobial effect of lactoferrin is also due to immu- Italy). Randomization was carried out using a ran- nomodulation and cytoplasmic membrane disruption dom number generator. Lactoferrin was adminis- of the target cell. Lactoferrin has an inhibitory effect on tered as vaginal tablets and each subject was lipopolysaccharide-induced production of inflamma- instructed to apply the tablet once a day, preferably tory cytokines [tumor necrosis factor-α,interleukin at night before going to bed. All the women under- (IL)-1β, IL-6, and IL-8 messenger RNA] and interferes went a complete assessment in three scheduled with nuclear factor-κB activation in monocytic cells appointments: at baseline (T0), on the 10th day of [26,27]. Additional functions of lactoferrin have been treatment (treatment: T1), and 2 weeks after the reported, such as neutrophil and macrophage activation end of lactoferrin administration (post-treatment: [28], regulation of specialization and function of lym- T2). Inclusion criteria were sexually active women phocytes [29], activation of natural killer cells [30], of reproductive age (18–45 years old) with regular control of oxidation injury [29], and down-modulation menstrual cycles and with symptomatic acute BV of ongoing immune-inflammatory responses during diagnosed according to Amsel’scriteria[33] and preterm delivery [31]. Moreover, lactoferrin exhibits a Nugent score [34]. The exclusion criteria were age synergistic effect with immunoglobulin A, lysozyme, < 18 years; Nugent score < 7; known active infec- antibiotics, and drugs, which helps in the eradication tion due to Chlamydia,yeasts,Neisseria gonor- of microorganisms [32]. Giunta and co-workers [31] rhoeae,orTrichomonas vaginalis; clinically demonstrated that oral administration of lactoferrin apparent herpes simplex infection or defined diag- 100 mg, twice a day for 1 month, led to the reduction nosed human papillomavirus, herpes simplex virus of IL-6 in cervicovaginal fluid and the normalization of type 2, or human immunodeficiency virus type 1 vaginal microbiota in women at preterm delivery risk. infection; pregnancy or breastfeeding; antibiotic, Otsuki and Imai [33] showed that, in women with a probiotic, or exogenous hormone treatments;