Complete Genome Sequence of Lactobacillus hilgardii LMG 7934, Carrying the Gene Encoding for the Novel PII-Like Protein PotN.

Lactic acid bacteria are widespread in various ecological niches with the excess of nutrients and have reduced capabilities to adapt to starvation. Among more than 280 Lactobacillus species known to the date, only five, including Lactobacillus hilgardii, carry in their genome the gene encoding for PII-like protein, one of the central regulators of cellular metabolism generally responding to energy- and carbon-nitrogen status in many free-living Bacteria, Archaea and in plant chloroplasts. In contrast to the classical PII encoding genes, in L. hilgardii genome the gene for PII homologue is located within the potABCD operon, encoding the ABC transporter for polyamines. Based on the unique genetic context and low sequence identity with genes of any other so-far characterized PII subfamilies, we termed this gene potN (Pot-protein, Nucleotide-binding). The second specific feature of L. hilgardii genome is that many genes encoding the proteins with similar function are present in two copies, while with low mutual identity. Thus, L. hilgardii LMG 7934 genome carries two genes of glutamine synthetase with 55% identity. One gene is located within classical glnRA operon with the gene of GlnR-like transcriptional regulator, while the second is monocistronic. Together with the relative large genome of L. hilgardii as compared to other Lactobacilli (2.771.862 bp vs ~ 2.2 Mbp in median), these data suggest significant re-arrangements of the genome and a wider range of adaptive capabilities of L. hilgardii in comparison to other bacteria of the genus Lactobacillus.

Increasing Susceptibility of Drug-Resistant Candida albicans to Fluconazole and Terbinafine by 2(5H)-Furanone Derivative.

The frequency of mycoses caused by drug-resistant fungal pathogen Candida albicans has increased drastically over the last two decades. The spread of drug-resistant strains, along with the limitations of currently available antifungals, complicates the management of fungal infections, thereby representing great challenges for clinical healthcare. Among various antimicrobial pharmacophores, 2(5H)-furanone derivatives have demonstrated antimicrobial, antifungal, and antibiofilm activities. In this study, we report the antifungal activity of the 2(5H)-furanone derivative F105, consisting of three pharmacophores, namely chlorinated 2(5H)-furanone, sulfonyl group, and l-menthol moiety. Although exhibiting moderate antifungal activity alone with the minimum inhibitory concentration (MIC) values of 32-256 µg/mL, F105 potentiates the activity of fluconazole and terbinafine with fractional inhibitory concentration index (FICI) values of 0.27-0.50. Thus, 16 µg/mL of F105 reduced the MICs of these antifungals against fluconazole-resistant C. albicans isolates four-fold, achieving similar values as for the intermediately susceptible phenotype. Confocal laser scanning microscopy revealed that the fluorescent 2(5H)-furanone derivative F145 was also able to penetrate through biofilms formed by C. albicans. Indeed, in the presence of F105, even sub-MIC concentrations of both fluconazole and terbinafine led to significant reduction of C. albicans CFUs in the mature biofilm. Thus, F105 appears to be a promising candidate for the development of novel antifungal agents as well as enhancers of current antifungal agents, particularly for the treatment of drug-resistant C. albicans infections.

Identification of Antimicrobial Peptides from Novel Lactobacillus fermentum Strain.

Antimicrobial peptides (AMPs) are natural antagonistic tools of many bacteria and are considered as attractive antimicrobial agents for the treatment of bacteria with multidrug resistance. Lactic acid bacteria from the gastrointestinal tract of animals and human produce various AMPs inhibiting the growth of pathogens. Here we report the isolation and identification of novel Lactobacillus fermentum strain HF-D1 from the human gut producing AMPs which prevents the growth of P. aeruginosa and S. marcescens. The active fraction of peptides was obtained from the culture liquid by precipitation at 80% saturation of ammonium sulphate. For peptides identification, the precipitate was treated with guanidine hydrochloride to desorb from proteins, separated with ultrafiltration on spin columns with 10,000 MWCO, desalted with a reversed-phase chromatography and subjected to LC-MS/MS analysis. The in silico analysis of the identified 1111 peptides by using ADAM, CAMPR3 and AMPA prediction servers led to identification of the linear peptide with highly probable antimicrobial activity and further investigation of its antibacterial activity mechanism is promising. By using the dereplication algorithm, the peptide highly similar to non-ribosomal cyclic AMPs originally isolated from Staphylococcus epidermidis has been identified. This indicates that L. fermentum HF-D1 represents a novel strain producing antimicrobial peptides targeting P. aeruginosa and S. marcescens.