Insight into the Lytic Functions of the Lactococcal Prophage TP712

The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria

Synergistic action of phage phiIPLA-RODI and lytic protein CHAPSH3b : a combination strategy to target Staphylococcus aureus biofilms

Staphylococcus aureus is considered a priority pathogen due to its increasing acquisition of antibiotic resistance determinants. Additionally, this microbe has the ability to form recalcitrant biofilms on different biotic and inert surfaces. In this context, bacteriophages and their derived lytic proteins may be a forward-looking strategy to help combat staphylococcal biofilms. However, these antimicrobials exhibit individual limitations that may be overcome by combining them with other compounds. This work investigates the combination of a phage-derived lytic protein, CHAPSH3b, and the virulent bacteriophage phiIPLA-RODI. The obtained results show the synergy between both antimicrobials for the treatment of 24-h-old S. aureus biofilms, with greater reductions in viable cell counts observed when phage and lysin are applied together compared to the individual treatments. Time-kill curves and confocal microscopy revealed that the fast antibacterial action of CHAPSH3b reduces the population up to 7 hours after initial exposure, which is subsequently followed by phage predation, limiting regrowth of the bacterial population. Moreover, at least 90% of bacteriophage insensitive mutants are susceptible to the lytic protein. Therefore, CHAPSH3b might help curtail the development of phage resistance during treatment. The combination of the lysin and phiIPLA-RODI also showed promising results in an ex vivo pig skin model of wound infection. Overall, the results of this study demonstrate that the combination of phage-derived lytic proteins and bacteriophages can be a viable strategy to develop improved antibiofilm products

Prevalence of submicroscopic malaria infection in immigrants living in Spain

BACKGROUND: The importance of submicroscopic malaria infections in high-transmission areas could contribute to maintain the parasite cycle. Regarding non-endemic areas, its importance remains barely understood because parasitaemia in these afebrile patients is usually below the detection limits for microscopy, hence molecular techniques are often needed for its diagnosis. In addition to this, the lack of standardized protocols for the screening of submicroscopic malaria in immigrants from endemic areas may underestimate the infection with Plasmodium spp. The aim of this study was to assess the prevalence of submicroscopic malaria in afebrile immigrants living in a non-endemic area. METHODS: A prospective, observational, multicentre study was conducted. Afebrile immigrants were included, microscopic observation of Giemsa-stained thin and thick blood smears, and two different molecular techniques detecting Plasmodium spp. were performed. Patients with submicroscopic malaria were defined as patients with negative blood smears and detection of DNA of Plasmodium spp. with one or both molecular techniques. Demographic, clinical, analytical and microbiological features were recorded and univariate analysis by subgroups was carried out with STATA v15. RESULTS: A total of 244 afebrile immigrants were included in the study. Of them, 14 had a submicroscopic malaria infection, yielding a prevalence of 5.7% (95% confidence interval 3.45-9.40). In 71.4% of the positive PCR/negative microscopy cases, Plasmodium falciparum alone was the main detected species (10 out of the 14 patients) and in 4 cases (28.6%) Plasmodium vivax or Plasmodium ovale were detected. One patient had a mixed infection including three different species. CONCLUSIONS: The prevalence of submicroscopic malaria in afebrile immigrants was similar to that previously described in Spain. Plasmodium vivax and P. ovale were detected in almost a third of the submicroscopic infections. Screening protocols for afebrile immigrants with molecular techniques could be useful for a proper management of these patients.This work was funded by projects PI14/01671, PI17/01791 and PI14CIII/00014, from the Instituto de Salud Carlos III (Ministry of Economy, Industry and Competitiveness) and cofounded by the European Regional Development Fund, and approved by the Ethics Committee of our Institution. There was no funding from the PCR manufacturers; they did not play any role in data analysis or in the reporting of the results.S

Use of Green Fluorescent Protein To Monitor Cell Envelope Stress in Lactococcus lactis▿ §

A Lactococcus lactis reporter system suitable to detect cell envelope stress in high-throughput settings was developed by fusing the CesR-regulated promoter of llmg0169 to the gfpuv gene. A dot blot assay allowed fast detection of green fluorescent protein (GFP) fluorescence even at low production levels. Unexpectedly, this promoter was also induced by mitomycin C via CesR

The behavior of Staphylococcus aureus dual-species biofilms treated with bacteriophage phiIPLA-RODI depends on the accompanying microorganism

Peer reviewe

A methyl esterase from Bifidobacterium longum subsp. longum reshapes the prebiotic properties of apple pectin by triggering differential modulatory capacity in faecal cultures

Abstract Pectin structures have received increasing attention as emergent prebiotics due to their capacity to promote beneficial intestinal bacteria. Yet the collective activity of gut bacterial communities to cooperatively metabolize structural variants of this substrate remains largely unknown. Herein, the characterization of a pectin methylesterase, BpeM, from Bifidobacterium longum subsp. longum, is reported. The purified enzyme was able to remove methyl groups from highly methoxylated apple pectin, and the mathematical modelling of its activity enabled to tightly control the reaction conditions to achieve predefined final degrees of methyl‐esterification in the resultant pectin. Demethylated pectin, generated by BpeM, exhibited differential fermentation patterns by gut microbial communities in in vitro mixed faecal cultures, promoting a stronger increase of bacterial genera associated with beneficial effects including Lactobacillus, Bifidobacterium and Collinsella. Our findings demonstrate that controlled pectin demethylation by the action of a B. longum esterase selectively modifies its prebiotic fermentation pattern, producing substrates that promote targeted bacterial groups more efficiently. This opens new possibilities to exploit biotechnological applications of enzymes from gut commensals to programme prebiotic properties

Design and Selection of Engineered Lytic Proteins With Staphylococcus aureus Decolonizing Activity

Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15–0.6 μM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 μM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 μg/cm(2)). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections

The human gallbladder microbiome is related to the physiological state and the biliary metabolic profile

Authors’ contributions IC, CMGB, JIR, SG, JMR, SD, and AM conceived and designed the study. NM, LR, IGD, JS, and ABC conducted the laboratory work. NM, AC, CMGB, and JIR collected and processed the biological samples. ABC developed the DNA extraction protocol. CM, MM, and MV performed the library preparation for NGS and the DNA sequencing. NM, CM, BS, MM, and MV analyzed the 16S data and the metagenomic data. JS, LR, JMR, and IC analyzed the NMR data. CMGB and JIR recruited the subjects. NM, MV, IC, SD, and AM prepared the figures and tables and wrote the manuscript. All authors read and approved the final manuscript.Background: The microbial populations of the human intestinal tract and their relationship to specific diseases have been extensively studied during the last decade. However, the characterization of the human bile microbiota as a whole has been hampered by difficulties in accessing biological samples and the lack of adequate methodologies to assess molecular studies. Although a few reports have described the biliary microbiota in some hepatobiliary diseases, the bile microbiota of healthy individuals has not been described. With this in mind, the goal of the present study was to generate fundamental knowledge on the composition and activity of the human bile microbiota, as well as establishing its potential relationship with human bile-related disorders. Results: Human bile samples from the gallbladder of individuals from a control group, without any record of hepatobiliary disorder, were obtained from liver donors during liver transplantation surgery. A bile DNA extraction method was optimized together with a quantitative PCR (qPCR) assay for determining the bacterial load. This allows the selection of samples to perform functional metagenomic analysis. Bile samples from the gallbladder of individuals suffering from lithiasis were collected during gallbladder resection and the microbial profiles assessed, using a 16S rRNA gene-based sequencing analysis, and compared with those of the control group. Additionally, the metabolic profile of the samples was analyzed by nuclear magnetic resonance (NMR). We detected, for the first time, bacterial communities in gallbladder samples of individuals without any hepatobiliary pathology. In the biliary microecosystem, the main bacterial phyla were represented by Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Significant differences in the relative abundance of different taxa of both groups were found. Sequences belonging to the family Propionibacteriaceae were more abundant in bile samples from control subjects; meanwhile, in patients with cholelithiasis members of the families Bacteroidaceae, Prevotellaceae, Porphyromonadaceae, and Veillonellaceae were more frequently detected. Furthermore, the metabolomics analysis showed that the two study groups have different metabolic profiles. Conclusions: Our results indicate that the gallbladder of human individuals, without diagnosed hepatobiliary pathology, harbors a microbial ecosystem that is described for the first time in this study. Its bacterial representatives and metabolites are different from those detected in people suffering from cholelithiasis. In this regard, since liver donors have been subjected to the specific conditions of the hospital's intensive care unit, including an antibiotic treatment, we must be cautious in stating that their bile samples contain a physiologically normal biliary microbiome. In any case, our results open up new possibilities to discover bacterial functions in a microbial ecosystem that has not previously been explored.Ministerio de Economía, Comercio y EmpresaSección Deptal. de Farmacia Galénica y Tecnología Alimentaria (Veterinaria)Fac. de VeterinariaTRUEpu