Bacteriocin Genes Research Articles

Antilisterial activity of raw sheep milk from two native Epirus breeds: Culture-dependent identification, bacteriocin gene detection and primary safety evaluation of the antagonistic LAB biota

Raw milk from native small ruminant breeds in Epirus, Greece, is a valuable natural source of autochthonous lactic acid bacteria (LAB) strains with superior biotechnological properties. In this study, two bulk milks (RM1, RM2) from two local sheep yards, intended for traditional Kefalotyri cheese production, were preselected for bacteriocin-like antilisterial activity by in vitro tests. Their antagonistic LAB biota was quantified followed by polyphasic (16S rRNA gene sequencing; IGS for Enterococcus; a multiplex-PCR for Leuconostoc) identification of 42 LAB (RM1/18; RM2/24) isolates further evaluated for bacteriocin encoding genes and primary safety traits. Representative isolates of the numerically dominant mesophilic LAB were Leuconostoc mesenteroides (10) in both RMs, Streptococcus parauberis (7) in RM2, and Lactococcus lactis (1) in RM1; the subdominant thermophilic LAB isolates were Enterococcus durans (8), E. faecium (6), E. faecalis (3), E. hirae (1), E. hermanniensis (1), Streptococcus lutetiensis (2), S. equinus (1) and S. gallolyticus (1). Based on their rpoB, araA, dsr and sorA profiles, six Ln. mesenteroides strains (8 isolates) were atypical lying between the subspecies mesenteroides and dextranicum, whereas two strains profiled with Ln. mesenteroides subsp. jonggajibkimchii that is first-time reported in Greek dairy food. Two RM1 E. faecium strain biotypes (3 isolates) showed strong, enterocin-mediated antilisterial activity due to entA/entB/entP possession. One E. durans from RM1 possessed entA and entP, while additional nine RM2 isolates of the E. faecium/durans group processed entA or entP singly. All showed direct (cell-associated) antilisterial activity only, as also both S. lutetiensis strains from RM2 did strongly. Desirably, no LAB isolate was β-hemolyrtic, or cytolysin-positive, or possessed vanA, vanB for vancomycin resistance, or agg, espA, hyl, and IS16 virulence genes. However, all three E. faecalis from RM2 possessed gelE and/or ace virulence genes. In conclusion, all Ln. mesenteroides strains, the two safe, enterocin A-B-P-producing E. faecium strains, and the two antilisterial S. lutetiensis strains should be validated further as potential costarter or adjunct cultures in Kefalotyri cheese. The prevalence of α-hemolytic pyogenic streptococci in raw milk, mainly S. parauberis in RM2, requires consideration in respect to subclinical mastitis in sheep and the farm hygiene overall.

Antimicrobial Peptides (Bacteriocins) Produced by Lactococcus lactis and Pediococcus pentosaceus Strains with Activity Against Clinical and Food-Borne Pathogens.

Bacteriocins are ribosomal-synthesized peptides with antimicrobial activity, produced by different groups of bacteria, including lactic acid bacteria (LAB). Most of the produced by LAB bacteriocins can be described with rather broad spectra of inhibition and they offer suggested applications in food preservation and pharmaceutical sector. Different LAB were isolated from fermented food products and fruits, obtained from the region of Pohang, Korea, and identified based on physiological, biochemical, and molecular methods. The promising isolates, Pediococcus pentosaceus 732, Lactococcus lactis 808, and Lactococcus lactis subsp. lactis 431, were identified based on biochemical, physiological, and biomolecular approaches, including 16S rRNA partial sequencing, and were evaluated for production of bacteriocin, including stability in presence of enzymes, chemicals, pH, and temperatures. Adherence properties for the expressed bacteriocins by P. pentosaceus 732, Lc. lactis 808, and Lc. lactis subsp. lactis 431 were evaluated at presence of selected chemicals, pH, and temperatures. The presence of bacteriocin genes in the strains was investigated and analyzed. The bacterial effect of bacteriocin produced by studied strains on Listeria spp. and Staphylococcus spp. has been shown for actively growing and stationary cells. Similar growth and bacteriocin production were observed when studied strains were cultured in MRS at 30°C or 37°C. The presence of nisin operon with some point mutations on the genomic DNA was recorded based on the performed PCR reactions targeting different genes associated with nisin expression for both lactococcal strains. Pediocin PA-1 operon was evaluated in a similar manner for P. pentosaceus 732.

Isolation of starch and protein degrading strain Bacillus subtilis FYZ1-3 from tobacco waste and genomic analysis of its tolerance to nicotine and inhibition of fungal growth.

Aerobic fermentation is an effective technique for the large-scale processing of tobacco waste. However, the specificity of the structure and composition of tobacco-derived organic matter and the toxic alkaloids in the material make it currently difficult to directly use microbial agents. In this study, a functional strain FYZ1-3 was isolated and screened from thermophilic phase samples of tobacco waste composting. This strain could withstand temperatures as high as 80°C and grow normally at 0.6% nicotine content. Furthermore, it had a strong decomposition capacity of tobacco-derived starch and protein, with amylase activity of 122.3 U/mL and protease activity and 52.3 U/mL, respectively. To further understand the mechanism of the metabolic transformation of the target, whole genome sequencing was used and the secondary metabolite gene cluster was predicted. The inhibitory effect of the strain on common tobacco fungi was verified using the plate confrontation and agar column methods. The results showed that the strain FYZ1-3 was Bacillus subtilis, with a genome size of 4.17 Mb and GC content of 43.68%; 4,338 coding genes were predicted. The genome was annotated and analyzed using multiple databases to determine its ability to efficiently degrade starch proteins at the molecular level. Moreover, 14 functional genes related to nicotine metabolism were identified, primarily located on the distinct genomic island of FYZ1-3, giving a speculation for its nicotine tolerance capability on the molecular mechanism. By mining the secondary metabolite gene cluster prediction, we found potential synthetic bacteriocin, antimicrobial peptide, and other gene clusters on its chromosome, which may have certain antibacterial properties. Further experiments confirmed that the FYZ1-3 strain was a potent growth inhibitor of Penicillium chrysogenum, Aspergillus sydowii, A. fumigatus, and Talaromyces funiculosus. The creation and industrial use of the functional strains obtained in this study provide a theoretical basis for its industrial use, where it would be of great significance to improve the utilization rate of tobacco waste.

Distribution and ecology of the generalist lactic acid bacterium Carnobacterium maltaromaticum in different freshwater habitats: Metabolic and antagonistic abilities.

We explored the distribution, metabolic and antagonistic activities of Carnobacterium maltaromaticum, isolated from freshwater locations in Denmark during winter or early spring. This species was widely distributed in such habitats although it was relatively rare in low pH locations. Isolates possessed a diverse metabolism, potentially enabling functional capacities independent of habitat. The intraspecies competition showed a relatively high degree of mostly low-intensity interactions, which overall were not correlated with phylogeny or location. Only a few isolates exhibited broad-spectrum inhibition activity, targeting species from other genera and families, including one isolate that exhibited a broad inhibitory activity due to H2 O2 production. Bioinformatic analyses revealed that the frequency of bacteriocinogenic systems was low, and only one unmodified bacteriocin, piscicolin 126, correlated with phenotypic antagonistic activity. Furthermore, most potential bacteriocin gene complexes were not complete. Overall, this study showed C. maltaromaticum to be a generalist (nomadic) species with a constant presence in freshwater habitats, especially those with pH values >5. General metabolic properties did not suggest a strong degree of adaptation to the freshwater environment, and bacteriocin-mediated antagonistic activities appeared to play a minimal ecological role.

Bifidobacterium longum subsp. infantis as widespread bacteriocin gene clusters carrier stands out among the Bifidobacterium.

Bifidobacterium is the dominant genus, particularly in the intestinal tract niche of healthy breast-fed infants, and many of these strains have been proven to elicit positive effects on infant development. In addition to its effective antimicrobial activity against detrimental microorganisms, it helps to improve the intestinal microbiota balance. The isolation and identification of bacteriocins from Bifidobacterium have been limited since the mid-1980s, leading to an underestimation of its ability for bacteriocin production. Here, we employed a silicon-based search strategy to mine 354 putative bacteriocin gene clusters (BGCs), most of which have never been reported, from the genomes of 759 Bifidobacterium strains distributed across 9 species. Consistent with previous reports, most Bifidobacterium strains did not carry or carry only a single BGC; however, Bifidobacterium longum subsp. infantis, in contrast to other Bifidobacterium species, carried numerous BGCs, including lanthipeptides, lasso peptides, thiopeptides, and class IId bacteriocins. The antimicrobial activity of the crude bacteriocins and transcription analysis confirmed its potential for bacteriocin biosynthesis. Additionally, we investigated the association of bacteriocins with the phylogenetic positions of their homologs from other genera and niches. In conclusion, this study re-examines a few Bifidobacterium species traditionally regarded as a poor source of bacteriocins. These bacteriocin genes impart a competitive advantage to Bifidobacterium in colonizing the infant intestinal tract. IMPORTANCE Development of the human gut microbiota commences from birth, with bifidobacteria being among the first colonizers of the newborn intestinal tract and dominating it for a considerable period. To date, the genetic basis for the successful adaptation of bifidobacteria to this particular niche remains unclear since studies have mainly focused on glycoside hydrolase and adhesion-related genes. Bacteriocins are competitive factors that help producers maintain colonization advantages without destroying the niche balance; however, they have rarely been reported in Bifidobacterium. The advancement in sequencing methods and bacteriocin databases enables the use of a silicon-based search strategy for the comprehensive and rapid re-evaluation of the bacteriocin distribution of Bifidobacterium. Our study revealed that B. infantis carries abundant bacteriocin biosynthetic gene clusters for the first time, presenting new evidence regarding the competitive interactions of Bifidobacterium in the infant intestinal tract.

Novel Strain Leuconostoc lactis DMLL10 from Traditional Korean Fermented Kimchi as a Starter Candidate for Fermented Foods.

Leuconostoc lactis strain DMLL10 was isolated from kimchi, a fermented vegetable, as a starter candidate through safety and technological assessments. Strain DMLL10 was susceptible to ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, and tetracycline. It did not show any hemolytic activity. Regarding its phenotypic results related to its safety properties, genomic analysis revealed that strain DMLL10 did not encode for any toxin genes such as hemolysin found in the same genus. It did not acquire antibiotic resistance genes either. Strain DMLL10 showed protease activity on agar containing NaCl up to 3%. The genome of DMLL10 encoded for protease genes and possessed genes associated with hetero- and homo-lactic fermentative pathways for lactate production. Finally, strain DMLL10 showed antibacterial activity against seven common foodborne pathogens, although bacteriocin genes were not identified from its genome. These results indicates that strain DMLL10 is a novel starter candidate with safety, enzyme activity, and bacteriocin activity. The complete genomic sequence of DMLL10 will contribute to our understanding of the genetic basis of probiotic properties and allow for assessment of the effectiveness of this strain as a starter or probiotic for use in the food industry.

Genomic Analysis of Bacteriocin-Producing Staphylococci: High Prevalence of Lanthipeptides and the Micrococcin P1 Biosynthetic Gene Clusters.

Bacteriocins are antimicrobial peptides produced by bacteria. This study aimed to in silico analyze the presence of bacteriocin gene clusters (BGCs) among the genomes of 22 commensal Staphylococcus isolates from different origins (environment/human/food/pet/wild animals) previously identified as bacteriocin producers. The resistome and plasmidome were studied in all isolates. Five types of BGC were detected in 18 genomes of the 22 bacteriocin-producing staphylococci included in this study: class I (Lanthipeptides), class II, circular bacteriocins, the non-ribosomal-peptide lugdunin and the thiopeptide micrococcin P1 (MP1). A high frequency of lanthipeptides was detected in this collection: BGC variants of BSA, bacCH91, and epilancin15X were identified in two Staphylococcus aureus and one Staphylococcus warneri isolates from food and wild animals. Moreover, two potentially new lanthipeptide-like BGCs with no identity to database entries were found in Staphylococcus epidermidis and Staphylococcus simulans from food and wild animal, respectively. Interestingly, four isolates (one S. aureus and one Staphylococcus hominis, environmental origin; two Staphylococcus sciuri, food) carried the MP1 BGC with differences to those previously described. On the other hand, seven of the 22 genomes (~32%) lacked known genes related with antibiotic or disinfectant-acquired resistance mechanisms. Moreover, the potential carriage of plasmids was evaluated, and several Rep-proteins were identified (~73% of strains). In conclusion, a wide variety of BGCs has been observed among the 22 genomes, and an interesting relationship between related Staphylococcus species and the type of bacteriocin has been revealed. Therefore, bacteriocin-producing Staphylococcus and especially coagulase-negative staphylococci (CoNS) can be considered good candidates as a source of novel bacteriocins.

Bioactivity Screening and Genomic Analysis Reveals Deep-Sea Fish Microbiome Isolates as Sources of Novel Antimicrobials.

With the increase in antimicrobial resistance and the subsequent demand for novel therapeutics, the deep-sea fish microbiome can be a relatively untapped source of antimicrobials, including bacteriocins. Previously, bacterial isolates were recovered from the gut of deep-sea fish sampled from the Atlantic Ocean.In this study, we used in vitro methods to screen a subset of these isolates for antimicrobial activity, and subsequently mined genomic DNA from isolates of interest for bacteriocin and other antimicrobial metabolite genes. We observed antimicrobial activity against foodborne pathogens, including Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis and Micrococcus luteus. In total, 147 candidate biosynthetic gene clusters were identified in the genomic sequences, including 35 bacteriocin/RiPP-like clusters. Other bioactive metabolite genes detected included non-ribosomal peptide synthases (NRPS), polyketide synthases (PKS; Types 1 and 3), beta-lactones and terpenes. Moreover, four unique bacteriocin gene clusters were annotated and shown to encode novel peptides: a class IIc bacteriocin, two class IId bacteriocins and a class I lanthipeptide (LanM subgroup). Our dual in vitro and in silico approach allowed for a more comprehensive understanding of the bacteriocinogenic potential of these deep-sea isolates and an insight into the antimicrobial molecules that they may produce.

Genomic insights into the safety and bacteriocinogenic potential of isolates from artisanal fermented milk Matsoni

Matsoni is a fermented milk common in the Caucasian region. In this study, whole-genome sequencing was performed with the aim of determining the taxonomic position of 25 previously selected candidate Matsoni starter cultures, identifying bacteriocin genes and prophages in their genomes, and evaluating their safety. In silico prediction of antibiotic resistance genes, virulence factors, bacteriocin genes and prophages was performed using dedicated databases and bioinformatics tools. With the exception of Enterococcus faecalis, which contained the mobile genetic element Tn916 with tetM and virulence factors, and Streptococcus thermophilus with mefE, the isolates were found to be safe for use as starter cultures. Thermophilin A, thermophilin 13, streptide and Blp genes were predicted in isolates of Streptococcus thermophilus, and genes for enterocins A, B, and X were found in Enterococcus faecium, but inhibition between strains was not observed. Several complete prophages were also detected in the genomes of Lactobacillus delbrueckii ssp. lactis. Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium. Some of these isolates are good candidates for customised autochthonous Matsoni starter cultures which are an alternative to the commercial starter cultures currently used in the industry.

Purification and characterization of a novel low-molecular-weight antimicrobial peptide produced by Lactiplantibacillus plantarum NMGL2

The present study aimed to purify and characterize a novel low-molecular-weight antimicrobial peptide (AMP) named as PNMGL2 produced by Lactiplantibacillus plantarum NMGL2. The AMP was effectively separated and purified by ethyl acetate extraction and DEAE-Sepharose anion exchange chromatography. Tricine-SDS-PAGE of the purified AMP showed a major protein band below 1.7 kDa, which was identified by MALDI-TOF MS to be a hexapeptide LNFLKK (761.95 Da), and structurally characterized to be combination of helixes and random coil by a PEP-FOLD 3 De novo approach. The antimicrobial activity of LNFLKK was confirmed by chemical synthesis of the peptide that showed clear inhibition (MIC 7.8 mg/mL) against both Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes), and Gram-negative bacteria (Enterobacter sakazakii, Escherichia coli and Shigella flexneri). PNMGL2 was pH resistant (pH 2–9), heat stable (121 °C, 30 min), and protease sensitive. Treatment of UV rays, sodium chloride and organic solvents did not decrease the activity. Sequencing of the whole genome of L. plantarum NMGL2 revealed presence of a bacteriocin gene cluster with two putative bacteriocin genes (ORF4 and ORF5) that were not expressed, confirming the significance of PNMGL2 contributing the antimicrobial activity of the strain. This study demonstrated the low-molecular-weight AMP that was uncharacterized in the relevant available databases, suggesting its potential application as a novel natural food preservative.

Nasopharyngeal competition dynamics are likely to be altered following vaccine introduction: bacteriocin prevalence and diversity among Icelandic and Kenyan pneumococci.

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit other bacteria in the surrounding environment. Streptococcus pneumoniae is a leading cause of disease worldwide and colonises the healthy human nasopharynx, where it competes for space and nutrients. Pneumococcal conjugate vaccines have reduced the incidence of disease, but they also restructure the bacterial population, and this restructuring likely alters the nasopharyngeal competition dynamics. Here, the distribution of bacteriocins was examined in over 5000 carriage and disease-causing pneumococci from Iceland and Kenya, recovered before and after the introduction of pneumococcal vaccination. Overall, up to eleven different bacteriocin gene clusters were identified per pneumococcus. Significant differences in the prevalence of bacteriocins were observed before and after vaccine introduction, and among carriage and disease-causing pneumococci, which were largely explained by the bacterial population structure. Genetically similar pneumococci generally harboured the same bacteriocins although sometimes different repertoires of bacteriocins were observed, which suggested that horizontal transfer of bacteriocin clusters had occurred. These findings demonstrated that vaccine-mediated changes in the pneumococcal population altered the prevalence and distribution of bacteriocins. The consequences of this for pneumococcal colonisation and disease remain to be determined.

Identification and characterization of a novel bacteriocin gene cluster in Lysinibacillus boronitolerans.

Although the antibiotics inhibit or kill pathogens, the abuse leads to the resistance formation and even "Super Bacteria." Therefore, it is urgent to explore the natural and safe alternatives such as bacteriocin. In this study, an uncharacterized bacteriocin gene cluster for Lysinibacillus boronitolerans was first predicted by genome sequencing and bioinformatics analysis, of which including twobiosynthetic genes, a regulatory gene, a transport-related gene, and six other genes. Subsequently, the 10.24-kb gene cluster was expressed in Escherichia coli BL21, and the lysate effectively inhibited the growths of pathogenic bacteria containing Bacillus pumilus, Bacillus velezensis, Pseudomonas syringae pv. tomato DC3000, and Xanthomonas axonopodis pv. manihotis. The antibacterial substance was purified by 70% ammonium sulfate precipitation and further identified by liquid chromatography-tandem mass spectrometry. The results showed that the antibacterial substance consisted of 44 amino acids and had 24.1% sequence identity with the cyanobacterin Piricyclamide 7005 E4 PirE4, a bacteriocin analogue. The minimal set of genes required for the biosynthesis of the antibacterial substance was determined by site-directed mutagenesis, suggesting both a transcriptional repressor and a phosphohydroxythreonine transaminase were essential. Subsequently, the evolution and conservation of the two proteins were analyzed among 22 Lysinibacillus species. Among them, the residues responsible for functions were identified. Collectively, our results set a solid foundation for investigation of the biosynthesis and application of bacteriocin.

Comparative genomics of food-derived probiotic Lactiplantibacillus plantarum K25 reveals its hidden potential, compactness, and efficiency.

This study aimed to investigate the intricate genetic makeup of the Lactiplantibacillus plantarum K25 strain by conducting a comprehensive analysis of comparative genomics. The results of our study demonstrate that the genome exhibits a high-level efficiency and compactness, comprising a total of 3,199 genes that encode proteins and a GC content of 43.38%. The present study elucidates the evolutionary lineage of Lactiplantibacillus plantarum strains through an analysis of the degree of gene order conservation and synteny across a range of strains, thereby underscoring their closely interrelated evolutionary trajectories. The identification of various genetic components in the K25 strain, such as bacteriocin gene clusters and prophage regions, highlights its potential utility in diverse domains, such as biotechnology and medicine. The distinctive genetic elements possess the potential to unveil innovative therapeutic and biotechnological remedies in future. This study provides a comprehensive analysis of the L. plantarum K25 strain, revealing its remarkable genomic potential and presenting novel prospects for utilizing its unique genetic features in diverse scientific fields. The present study contributes to the existing literature on Lactiplantibacillus plantarum and sets the stage for prospective investigations and practical implementations that leverage the exceptional genetic characteristics of this adap organism.

Complete Genome Sequencing and Bacteriocin Functional Characterization of Pediococcus ethanolidurans CP201 from Daqu.

This study aims to sequence the whole genome of Pediococcus ethanolidurans CP201 isolated from Daqu and determine the anti-corrosion ability of bacteriocins on chicken breast. The whole genome sequence information of P. ethanolidurans CP201 was analyzed, and its gene structure and function were explored. It was found that gene1164 had annotations in the NR, Pfam, and Swiss-Prot databases, and was related to bacteriocins. The exogenous expression of the bacteriocin gene Pediocin PE-201 was analyzed based on the pET-21b vector and the host BL21, and the corresponding bacteriocin was successfully expressed under the induction of IPTG. After purification by NI-NTA column, enterokinase treatment, membrane dialysis concentration treatment, and SDS-PAGE electrophoresis, the molecular weight was about 6.5kDa and the purity was above 90%. By applying different concentrations of bacteriocin to chicken breast with different levels of contamination, the control of pathogenic bacteria, the ordinary contamination level (OC) group, and the high contamination level (MC) group could be completely achieved with 25mg/L bacteriocin. In conclusion, the bacteriocin produced by the newly isolated CP201 can be applied to the preservation of meat products to prevent the risk of food-borne diseases.

Metagenome-mining indicates an association between bacteriocin presence and strain diversity in the infant gut

BackgroundOur knowledge about the ecological role of bacterial antimicrobial peptides (bacteriocins) in the human gut is limited, particularly in relation to their role in the diversification of the gut microbiota during early life. The aim of this paper was therefore to address associations between bacteriocins and bacterial diversity in the human gut microbiota. To investigate this, we did an extensive screening of 2564 healthy human gut metagenomes for the presence of predicted bacteriocin-encoding genes, comparing bacteriocin gene presence to strain diversity and age.ResultsWe found that the abundance of bacteriocin genes was significantly higher in infant-like metagenomes (< 2 years) compared to adult-like metagenomes (2–107 years). By comparing infant-like metagenomes with and without a given bacteriocin, we found that bacteriocin presence was associated with increased strain diversities.ConclusionsOur findings indicate that bacteriocins may play a role in the strain diversification during the infant gut microbiota establishment.

Microbial community and antibiotic resistance gene distribution in food waste, anaerobic digestate, and paddy soil

The study assessed the occurrence and distribution of microbial community and antibiotic resistance genes (ARGs) in food waste, anaerobic digestate, and paddy soil samples, and revealed the potential hosts of ARGs and factors influencing their distribution. A total of 24 bacterial phyla were identified, of which 16 were shared by all samples, with Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria accounting for 65.9–92.3 % of the total bacterial community. Firmicutes was the most abundant bacteria in food waste and digestate samples, accounting for 33–83 % of the total microbial community. However, in paddy soil samples with digestate, Proteobacteria had the highest relative abundance of 38–60 %. Further, 22 ARGs were detected in food waste and digestate samples, with multidrug, macrolide-lincosamide-streptogramin (MLS), bacitracin, aminoglycoside, tetracycline, vancomycin, sulfonamide, and rifamycin resistance genes being the most abundant and shared by all samples. The highest total relative abundance of ARGs in food waste, digestate, and soil without and with digestate was detected in samples from January 2020, May 2020, October 2019, and May 2020, respectively. The MLS, vancomycin, tetracycline, aminoglycoside, and sulfonamide resistance genes had higher relative abundance in food waste and anaerobic digestate samples, whereas multidrug, bacteriocin, quinolone, and rifampin resistance genes were more abundant in paddy soil samples. Redundancy analysis demonstrated that aminoglycoside, tetracycline, sulfonamide, and rifamycin resistance genes were positively correlated with total ammonia nitrogen and pH of food waste and digestate samples. Vancomycin, multidrug, bacitracin, and fosmidomycin resistance genes had positive correlations with potassium, moisture, and organic matter in soil samples. The co-occurrence of ARG subtypes with bacterial genera was investigated using network analysis. Actinobacteria, Proteobacteria, Bacteroidetes, and Acidobacteria were identified as potential hosts of multidrug resistance genes.

Probiotic Properties and Antioxidant Activity In Vitro of Lactic Acid Bacteria.

The properties of probiotics such as lactic acid bacteria (LAB) have been widely studied over the last decades. In the present study, four different LAB species, namely Lactobacillus gasseri ATCC 33323, Lacticaseibacillus rhamnosus GG ATCC 53103, Levilactobacillus brevis ATCC 8287, and Lactiplantibacillus plantarum ATCC 14917, were investigated in order to determine their ability to survive in the human gut. They were evaluated based on their tolerance to acids, resistance to simulated gastrointestinal conditions, antibiotic resistance, and the identification of genes encoding bacteriocin production. All four tested strains demonstrated high resistance to simulated gastric juice after 3 h, and the viable counts revealed declines in cell concentrations of less than 1 log cycle. L. plantarum showed the highest level of survival in the human gut, with counts of 7.09 log CFU/mL. For the species L. rhamnosus and L. brevis, the values were 6.97 and 6.52, respectively. L. gasseri, after 12 h, showed a 3.96 log cycle drop in viable counts. None of the evaluated strains inhibited resistance to ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, or chloramphenicol. With regard to bacteriocin genes, the Pediocin PA gene was identified in Lactiplantibacillus plantarum ATCC 14917, Lacticaseibacillus rhamnosus GG ATCC 53103, and Lactobacillus gasseri ATCC 33323. The PlnEF gene was detected in Lactiplantibacillus plantarum ATCC 14917 and Lacticaseibacillus rhamnosus GG ATCC 53103. The Brevicin 174A and PlnA genes were not detected in any bacteria. Moreover, the potential antioxidant activity of LAB's metabolites was evaluated. At the same time, the possible antioxidant activity of metabolites of LAB was first tested using the free radical DDPH• (a, a-Diphenyl-β-Picrylhydrazyl) and then evaluated with regard to their radical scavenging activity and inhibition against peroxyl radical induced DNA scission. All strains showed antioxidant activity; however, the best antioxidant activity was achieved by L. brevis (94.47%) and L. gasseri (91.29%) at 210 min. This study provides a comprehensive approach to the action of these LAB and their use in the food industry.

Comprehensive Analysis of Bacteriocins Produced by the Hypermucoviscous Klebsiella pneumoniae Species Complex.

Klebsiella pneumoniae produces several kinds of bacteriocins that have antimicrobial effects against closely related species, but few studies have comprehensively reported bacteriocin distribution among the Klebsiella population. In this study, we identified bacteriocin genes in 180 K. pneumoniae species complex genomes, including 170 hypermucoviscous isolates, and investigated the antibacterial activity against 50 strains, including antimicrobial-resistant organisms, belonging to multiple species, namely, Klebsiella spp., Escherichia coli, Pseudomonas spp., Acinetobacter spp., Enterobacter cloacae, Stenotrophomonas maltophilia, Chryseobacterium indologenes, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus mutans. Our study determined that 32.8% (59/180) of isolates carried at least one bacteriocin type. Different types of bacteriocin were usually present in different specific sequence types (STs); meanwhile, bacteriocins were not detected in certain STs. Microcin E492 was the most prevalent bacteriocin (14.4%), mostly in ST23 isolates, and displayed a wide spectrum of activity, including against Klebsiella spp., E. coli, Pseudomonas spp., and Acinetobacter spp. Cloacin-like bacteriocin was detected in 7.2% of strains, all of which were non-ST23 isolates, and exhibited inhibitory activity against closely related species, mainly Klebsiella spp. Klebicin B-like bacteriocin was detected at a rate of 9.4%, although 82.4% of these strains carried a disrupted bacteriocin gene, and an inhibitory effect could not be observed from the intact-gene-carrying isolates. Other bacteriocins, such as microcin S-like, microcin B17, and klebicin C-like, were detected at lower rates and had limited inhibitory activity. Our findings suggested that Klebsiella strains that carry different bacteriocin types may affect the composition of the surrounding bacterial community. IMPORTANCE Klebsiella pneumoniae is a Gram-negative commensal bacterium that asymptomatically colonizes human mucosal membranes, such as the intestinal tract, but it is also a leading cause of health care- and community-associated infections. Additionally, multidrug-resistant K. pneumoniae has been continuously evolving, which significantly challenges the available chemotherapeutic treatment for its infections. K. pneumoniae produces several kinds of antimicrobial peptides known as bacteriocins, which have antibacterial activity against closely related species. This work was the first comprehensive report of bacteriocin distribution among the hypermucoviscous K. pneumoniae species complex population and the inhibitory activity of each bacteriocin type against various species, including multidrug-resistant strains. Our findings provide a foundation for future studies on the K. pneumoniae species complex, including studies on the competition within the microflora and the potential applications of bacteriocins in treating multidrug-resistant bacteria.

Thermophilin 13: In Silico Analysis Provides New Insight in Genes Involved in Bacteriocin Production.

Bacteriocins are a large family of ribosomally synthesised proteinaceous toxins that are produced by bacteria and archaea and have antimicrobial activity against closely related species to the producer strain. Antimicrobial proteinaceous compounds are associated with a wide range of applications, including as a pathogen inhibitor in food and medical use. Among the several lactic acid bacteria (LAB) commonly used in fresh and fermented food preservation, Streptococcus thermophilus is well known for its importance as a starter culture for yoghurt and cheese. Previous studies described the bacteriocin thermophilin 13 exclusively in S. thermophilus SFi13 and the genes encoding its production as an operon consisting of two genes (thmA and thmB). However, the majority of bacteriocins possess a complex production system, which involves several genes encoding dedicated proteins with relatively specific functions. Up to now, far too little attention has been paid to the genes involved in the synthesis, regulation and expression of thermophilin 13. The aim of the present study, using in silico gene mining, was to investigate the presence of a regulation system involved in thermophilin 13 production. Results revealed the dedicated putative bacteriocin gene cluster (PBGC), which shows high similarity with the class IIb bacteriocins genes. This newly revealed PBGC, which was also found within various strains of Streptococcus thermophilus, provides a new perspective and insights into understanding the mechanisms implicated in the production of thermophilin 13.

Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants.

Lactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642kDa truncated form of LBU bacteriocin consisting of 31N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25-43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1-24, the truncation at position 31 is predicted to change the structure within aa 15-31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively.