Elemental sulfur enhances the anti-fungal effect of Lacticaseibacillus rhamnosus Lcr35.

Lacticaseibacillus rhamnosus Lcr35 is a well-known bacterial strain whose efficiency in preventing recurrent vulvovaginal candidiasis has been largely demonstrated in clinical trials. The presence of sodium thiosulfate (STS) has been shown to enhance its ability to inhibit the growth of Candida albicans strains. In this study, we confirmed that Lcr35 has a fungicidal effect not only on the planktonic form of C. albicans but also on other life forms such as hypha and biofilm. Transcriptomic analysis showed that the presence of C. albicans induced a metabolic adaptation of Lcr35 potentially associated with a competitive advantage over yeast cells. However, STS alone had no impact on the global gene expression of Lcr35, which is not in favor of the involvement of an enzymatic transformation of STS. Comparative HPLC and gas chromatography-mass spectrometry analysis of the organic phase from cell-free supernatant (CFS) fractions obtained from Lcr35 cultures performed in the presence and absence of STS identified elemental sulfur (S0) in the samples initially containing STS. In addition, the anti-Candida activity of CFS from STS-containing cultures was shown to be pH-dependent and occurred at acidic pH lower than 5. We next investigated the antifungal activity of lactic acid and acetic acid, the two main organic acids produced by lactobacilli. The two molecules affected the viability of C. albicans but only at pH 3.5 and in a dose-dependent manner, an antifungal effect that was enhanced in samples containing STS in which the thiosulfate was decomposed into S0. In conclusion, the use of STS as an excipient in the manufacturing process of Lcr35 exerted a dual action since the production of organic acids by Lcr35 facilitates the decomposition of thiosulfate into S0, thereby enhancing the bacteria's own anti-fungal effect.

Role of Klebsiella pneumoniae Type VI secretion system (T6SS) in long-term gastrointestinal colonization.

Type VI secretion systems (T6SS), recently described in hypervirulent K. pneumoniae (hvKp) strains, are involved in bacterial warfare but their role in classical clinical strains (cKp) has been little investigated. In silico analysis indicated the presence of T6SS clusters (from zero to four), irrespective of the strains origin or virulence, with a high prevalence in the K. pneumoniae species (98%). In the strain CH1157, two T6SS-apparented pathogenicity islands were detected, T6SS-1 and -2, harboring a phospholipase-encoding gene (tle1) and a potential new effector-encoding gene named tke (Type VI Klebsiella effector). Tle1 expression in Escherichia coli periplasm affected cell membrane permeability. T6SS-1 isogenic mutants colonized the highest gastrointestinal tract of mice less efficiently than their parental strain, at long term. Comparative analysis of faecal 16S sequences indicated that T6SS-1 impaired the microbiota richness and its resilience capacity. Oscillospiraceae family members could be specific competitors for the long-term gut establishment of K. pneumoniae.

Identification of sulfur components enhancing the anti-Candida effect of Lactobacillus rhamnosus Lcr35.

GYNOPHILUS (Lcr REGENERANS) is a live biotherapeutic product (LBP) aimed at restoring the vaginal microbiome and contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35. In this study, the LBP formulation and manufacturing process significantly enhanced the anti-Candida activity of L. rhamnosus Lcr35, with a complete loss of viability of the yeast after 48 h of coincubation. Sodium thiosulfate (STS), one excipient of the product, was used as a potentiator of the anti-Candida spp. activity of Lactobacilli. This contact-independent phenomenon induced fungal cell disturbances, as observed by electron microscopy observations. Nonverbal sensory experiments showed clear odor dissimilarities between cocultures of L. rhamnosus Lcr35 and C. albicans in the presence and absence of STS, suggesting an impact of odor-active metabolites. A volatolomic approach allowed the identification of six odor-active compounds, including one sulfur compound that was identified as S-methyl thioacetate (MTA). MTA was associated with the antifungal effect of Lcr35, and its functional link was established in vitro. We show for the first time that the LBP GYNOPHILUS, which is a highly active product in the reduction of vulvovaginal candidiasis, requires the presence of a sulfur compound to fully achieve its antifungal effect.

A mouse ear skin model to study the dynamics of innate immune responses against Staphylococcus aureus biofilms.

BACKGROUND: Staphylococcus aureus is a human pathogen that is a common cause of nosocomial infections and infections on indwelling medical devices, mainly due to its ability to shift between the planktonic and the biofilm/sessile lifestyle. Biofilm infections present a serious problem in human medicine as they often lead to bacterial persistence and thus to chronic infections. The immune responses elicited by biofilms have been described as specific and ineffective. In the few experiments performed in vivo, the importance of neutrophils and macrophages as a first line of defence against biofilm infections was clearly established. However, the bilateral interactions between biofilms and myeloid cells remain poorly studied and analysis of the dynamic processes at the cellular level in tissues inoculated with biofilm bacteria is still an unexplored field. It is urgent, therefore, to develop biologically sound experimental approaches in vivo designed to extract specific immune signatures from the planktonic and biofilm forms of bacteria. RESULTS: We propose an in vivo transgenic mouse model, used in conjunction with intravital confocal microscopy to study the dynamics of host inflammatory responses to bacteria. Culture conditions were created to prepare calibrated inocula of fluorescent planktonic and biofilm forms of bacteria. A confocal imaging acquisition and analysis protocol was then drawn up to study the recruitment of innate immune cells in the skin of LysM-EGFP transgenic mice. Using the mouse ear pinna model, we showed that inflammatory responses to S. aureus can be quantified over time and that the dynamics of innate immune cells after injection of either the planktonic or biofilm form can be characterized. First results showed that the ability of phagocytic cells to infiltrate the injection site and their motility is not the same in planktonic and biofilm forms of bacteria despite the cells being considerably recruited in both cases. CONCLUSION: We developed a mouse model of infection to compare the dynamics of the inflammatory responses to planktonic and biofilm bacteria at the tissue and cellular levels. The mouse ear pinna model is a powerful imaging system to analyse the mechanisms of biofilm tolerance to immune attacks.

Ciprofloxacin residue and antibiotic-resistant biofilm bacteria in hospital effluent.

Discharge of antimicrobial residues and resistant bacteria in hospital effluents is supposed to have strong impacts on the spread of antibiotic resistant bacteria in the environment. This study aimed to characterize the effluents of the Gabriel Montpied teaching hospital, Clermont-Ferrand, France, by simultaneously measuring the concentration of ciprofloxacin and of biological indicators resistant to this molecule in biofilms formed in the hospital effluent and by comparing these data to ciprofloxacin consumption and resistant bacterial isolates of the hospital. Determination of the measured environmental concentration of ciprofloxacin by spot sampling and polar organic chemical integrative (POCIS) sampling over 2 weeks, and comparison with predicted environmental concentrations produced a hazard quotient >1, indicating a potential ecotoxicological risk. A negative impact was also observed with whole hospital effluent samples using the Tetrahymena pyriformis biological model. During the same period, biofilms were formed within the hospital effluent, and analysis of ciprofloxacin-resistant isolates indicated that Gamma-Proteobacteria were numerous, predominantly Aeromonadaceae (69.56%) and Enterobacteriaceae (22.61%). Among the 115 isolates collected, plasmid-mediated fluoroquinolone-resistant genes were detected, with mostly aac(6')-lb-cr and qnrS. In addition, 60% of the isolates were resistant to up to six antibiotics, including molecules mostly used in the hospital (aminosides and third-generation cephalosporins). In parallel, 1247 bacteria isolated from hospitalized patients and resistant to at least one of the fluoroquinolones were collected. Only 5 of the 14 species identified in the effluent biofilm were also found in the clinical isolates, but PFGE typing of the Gram-negative isolates found in both compartments showed there was no clonality among the strains. Altogether, these data confirm the role of hospital loads as sources of pollution for wastewater and question the role of environmental biofilms communities as efficient shelters for hospital-released resistance genes.

A tripartite efflux pump involved in gastrointestinal colonization by Klebsiella pneumoniae confers a tolerance response to inorganic acid.

The colonization of the gastrointestinal tract of patients by the opportunistic gram-negative bacillus Klebsiella pneumoniae generally occurs prior to the development of nosocomial infections. Mutant strain C-81 was isolated owing to its reduced capacity to colonize the digestive tract in a murine model following transposon mutagenesis (N. Maroncle, D. Balestrino, C. Rich, and C. Forestier, Infect. Immun. 70:4729-4734, 2002). Nucleotide sequence analysis showed that the transposon had inserted into the first open reading frame, eefA, of a three-gene locus (eefABC) whose homologue encodes a tripartite efflux pump in Enterobacter aerogenes (M. Masi, J. M. Pages, C. Villard, and E. Pradel, J. Bacteriol. 187:3894-3897, 2005), and this operon includes an additional short (183-bp) potential open reading frame, eefX, upstream of eefA. In vivo assays showed that a DeltaeefA isogenic mutant strain normally colonized the gastrointestinal tract in single-strain tests but was significantly impaired in competition against wild-type strain LM21. Although the cecum was the compartment with the highest number of CFU, the DeltaeefA mutant also was detected in the stomach in numbers smaller than those of the wild-type strain. The expression of this potential efflux pump could not be linked to any antimicrobial drug resistance phenotype, but it conferred on the bacteria an acid tolerance response to inorganic acid. The expression of the eef promoter region, measured via a lacZ reporter construction, was slightly induced by an acidic environment and also by hyperosmolarity but not by the presence of bile salts. These results suggest that an efflux pump can confer measurable ecological benefits on K. pneumoniae in an environment with high competition potential.