Rhizosphere microbiomes of resurrection plants Ramonda serbica and R. nathaliae: comparative analysis and search for bacteria mitigating drought stress in wheat (Triticum aestivum L.).

Rhizosphere microbial communities play an important role in maintaining the health and productivity of the plant host. The rhizobacteria Pseudomonas putida P2 of Ramonda serbica and Bacillus cereus P5 of R. nathaliae were selected for treatment of the Belija wheat cultivar because of their plant growth-promoting (PGP) properties. Compared to the non-treated drought-stressed plants, the plants treated with rhizobacteria showed increased activity of the two major antioxidant enzymes, superoxide dismutase, and ascorbate peroxidase. Plants treated with the B. cereus P5 strain exhibited higher proline content under drought stress, suggesting that proline accumulation depends on the relative water content (RWC) status of the plants studied. Inoculation of wheat seeds with the P. putida P2 strain improved water status by increasing RWC and alleviating oxidative stress by reducing H2O2 and malondialdehyde concentrations in plants exposed to severe drought, possibly also helping plants to overcome drought through its 1-aminocyclopropane-1-carboxylic acid deaminase activity. Analysis of data from Next Generation sequencing (NGS) revealed that the dominant bacterial taxa in the rhizosphere of resurrection plants R. serbica and R. nathaliae were extremophilic, thermotolerant, Vicinamibacter silvestris, Chthoniobacter flavus, and Gaiella occulta. From the fungi detected Penicillium was the most abundant in both samples, while Fusarium and Mucor were present only in the rhizosphere of R. serbica and the entomopathogenic fungi Metarhizium, and Tolypocladiumu only in the rhizosphere of R. nathaliae. The fungal communities varied among plants, suggesting a stronger environmental influence than plant species. Our study demonstrates the importance of in vivo experiments to confirm the properties of PGP bacteria and indicates that the rhizosphere of resurrection plants is a valuable source of unique microorganisms that can be used to improve the drought stress tolerance of crops.

Culture-Dependent and Metabarcoding Characterization of the Sugar Beet (Beta vulgaris L.) Microbiome for High-Yield Isolation of Bacteria with Plant Growth-Promoting Traits.

The diversity of plant-associated bacteria is vast and can be determined by 16S rRNA gene metabarcoding. Fewer of them have plant-beneficial properties. To harness their benefits for plants, we must isolate them. This study aimed to check whether 16S rRNA gene metabarcoding has predictive power in identifying the majority of known bacteria with plant-beneficial traits that can be isolated from the sugar beet (Beta vulgaris L.) microbiome. Rhizosphere and phyllosphere samples collected during one season at different stages of plant development were analyzed. Bacteria were isolated on rich unselective media and plant-based media enriched with sugar beet leaves or rhizosphere extracts. The isolates were identified by sequencing the 16S rRNA gene and tested in vitro for their plant-beneficial properties (stimulation of germination; exopolysaccharide, siderophore, and HCN production; phosphate solubilization; and activity against sugar beet pathogens). The highest number of co-occurring beneficial traits was eight, found in isolates of five species: Acinetobacter calcoaceticus, Bacillus australimaris, B. pumilus, Enterobacter ludwiigi, and Pantoea ananatis. These species were not detected by metabarcoding and have not previously been described as plant-beneficial inhabitants of sugar beets. Thus, our findings point out the necessity of a culture-dependent microbiome analysis and advocate for low-nutrient plant-based media for high-yield isolation of plant-beneficial taxa with multiple beneficial traits. A culture-dependent and -independent approach is required for community diversity assessment. Still, isolation on plant-based media is the best approach to select isolates for potential use as biofertilizers and biopesticides in sugar beet cultivation.

Genome Analysis of Two Pseudomonas syringae pv. aptata Strains with Different Virulence Capacity Isolated from Sugar Beet: Features of Successful Pathogenicity in the Phyllosphere Microbiome.

Members of the Pseudomonas syringae species complex are heterogeneous bacteria that are the most abundant bacterial plant pathogens in the plant phyllosphere, with strong abilities to exist on and infect different plant hosts and survive in/outside agroecosystems. In this study, the draft genome sequences of two pathogenic P. syringae pv. aptata strains with different in planta virulence capacities isolated from the phyllosphere of infected sugar beet were analyzed to evaluate putative features of survival strategies and to determine the pathogenic potential of the strains. The draft genomes of P. syringae pv. aptata strains P16 and P21 are 5,974,057 bp and 6,353,752 bp in size, have GC contents of 59.03% and 58.77%, respectively, and contain 3,439 and 3,536 protein-coding sequences, respectively. For both average nucleotide identity and pangenome analysis, P16 and P21 largely clustered with other pv. aptata strains from the same isolation source. We found differences in the repertoire of effectors of the type III secretion system among all 102 selected strains, suggesting that the type III secretion system is a critical factor in the different virulent phenotypes of P. syringae pv. aptata. During genome analysis of the highly virulent strain P21, we discovered genes for T3SS effectors (AvrRpm1, HopAW1, and HopAU1) that were not previously found in genomes of P. syringae pv. aptata. We also identified coding sequences for pantothenate kinase, VapC endonuclease, phospholipase, and pectate lyase in both genomes, which may represent novel effectors of the type III secretion system. IMPORTANCE Genome analysis has an enormous effect on understanding the life strategies of plant pathogens. Comparing similarities with pathogens involved in other epidemics could elucidate the pathogen life cycle when a new outbreak happens. This study represents the first in-depth genome analysis of Pseudomonas syringae pv. aptata, the causative agent of leaf spot disease of sugar beet. Despite the increasing number of disease reports in recent years worldwide, there is still a lack of information about the genomic features, epidemiology, and pathogenic life strategies of this particular pathogen. Our findings provide advances in disease etiology (especially T3SS effector repertoire) and elucidate the role of environmental adaptations required for prevalence in the pathobiome of the sugar beet. From the perspective of the very heterogeneous P. syringae species complex, this type of analysis has specific importance in reporting the characteristics of individual strains.

Natural bacterial isolates as an inexhaustible source of new bacteriocins.

Microorganisms isolated from various traditionally fermented food products prepared in households without commercial starter cultures are designated as natural isolates. In addition, this term is also used for microorganisms collected from various natural habitats or products (silage, soil, manure, plant and animal material, etc.) that do not contain any commercial starters or bacterial formulations. They are characterized by unique traits that are the result of the selective pressure of environmental conditions, as well as interactions with other organisms. The synthesis of antimicrobial molecules, including bacteriocins, is an evolutionary advantage and an adaptive feature that sets them apart from other microorganisms from a common environment. This review aims to underline the knowledge of bacteriocins produced by natural isolates, with a particular emphasis on the most common location of their genes and operons, plasmids, and the importance of the relationship between the plasmidome and the adaptive potential of the isolate. Applications of bacteriocins, ranging from natural food preservatives to supplements and drugs in pharmacology and medicine, will also be addressed. The latest challenges faced by researchers in isolating new natural isolates with desired characteristics will be discussed, as well as the production of new antimicrobials, nearly one century since the first discovery of colicins in 1925. KEY POINTS: • Natural bacterial isolates harbor unique properties shaped by diverse interactions. • Horizontal gene transfer enables constant engineering of new antimicrobials. • Fermented food products are important source of bacteriocin-producing natural isolates.

Diversity of non-starter lactic acid bacteria in autochthonous dairy products from Western Balkan Countries - Technological and probiotic properties.

The aim of this review was to summarize the data regarding diversity of non-starter lactic acid bacteria (NSLAB) isolated from various artisanal dairy products manufactured in Western Balkan Countries. The dairy products examined were manufactured from raw cow's, sheep's or goat's milk or mixed milk, in the traditional way without the addition of commercial starter cultures. Dairy products such as white brined cheese, fresh cheese, hard cheese, yogurt, sour cream and kajmak were sampled in the households of Serbia, Croatia, Slovenia, Bosnia and Herzegovina, Montenegro, and North Macedonia. It has been established that the diversity of lactic acid bacteria (LAB) from raw milk artisanal dairy products is extensive. In the reviewed literature, 28 LAB species and a large number of strains belonging to the Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Pediococcus, Leuconostoc and Weissella genera were isolated from various dairy products. Over 3000 LAB strains were obtained and characterized for their technological and probiotic properties including: acidification and coagulation of milk, production of aromatic compounds, proteolytic activity, bacteriocins production and competitive exclusion of pathogens, production of exopolysaccharides, aggregation ability and immunomodulatory effect. Results show that many of the isolated NSLAB strains had one, two or more of the properties mentioned. The data presented emphasize the importance of artisanal products as a valuable source of NSLAB with unique technological and probiotic features important both as a base for scientific research as well as for designing novel starter cultures for functional dairy food.

Phenol Removal Capacity of the Common Duckweed (Lemna minor L.) and Six Phenol-Resistant Bacterial Strains From Its Rhizosphere: In Vitro Evaluation at High Phenol Concentrations.

The main topic of this study is the bioremediation potential of the common duckweed, Lemna minor L., and selected rhizospheric bacterial strains in removing phenol from aqueous environments at extremely high initial phenol concentrations. To that end, fluorescence microscopy, MIC tests, biofilm formation, the phenol removal test (4-AAP method), the Salkowski essay, and studies of multiplication rates of sterile and inoculated duckweed in MS medium with phenol (200, 500, 750, and 1000 mg L-1) were conducted. Out of seven bacterial strains, six were identified as epiphytes or endophytes that efficiently removed phenol. The phenol removal experiment showed that the bacteria/duckweed system was more efficient during the first 24 h compared to the sterile duckweed control group. At the end of this experiment, almost 90% of the initial phenol concentration was removed by both groups, respectively. The bacteria stimulated the duckweed multiplication even at a high bacterial population density (>105 CFU mL-1) over a prolonged period of time (14 days). All bacterial strains were sensitive to all the applied antibiotics and formed biofilms in vitro. The dual bacteria/duckweed system, especially the one containing strain 43-Hafnia paralvei C32-106/3, Accession No. MF526939, had a number of characteristics that are advantageous in bioremediation, such as high phenol removal efficiency, biofilm formation, safety (antibiotic sensitivity), and stimulation of duckweed multiplication.

Functional Characterization of the Lactolisterin BU Gene Cluster of Lactococcus lactis subsp. lactis BGBU1-4.

The gene cluster responsible for the production of the aureocin A53-like bacteriocin, lactolisterin BU, is located on plasmid pBU6 in Lactococcus lactis subsp. lactis BGBU1-4. Heterologous expression of pBU6 confirmed that production and limited immunity to lactolisterin BU were provided by the plasmid. Comparative analysis of aureocin A53-like operons revealed that the structural genes shared a low level of identity, while other genes were without homology, indicating a different origin. Subcloning and expression of genes located downstream of the structural gene, lliBU, revealed that the lactolisterin BU cluster consists of four genes: the structural gene lliBU, the abcT gene encoding an ABC transporter, the accL gene encoding an accessory protein and the immL gene which provides limited immunity to lactolisterin BU. Reverse transcription analysis revealed that all genes were transcribed as one polycistronic mRNA. Attempts to split the lactolisterin BU operon, even when both parts were under control of the PlliBU promoter, were unsuccessful indicating that expression of lactolisterin BU is probably precisely regulated at the translational level by translational coupling and is possible only when all genes of the operon are in cis constellation. Two ρ-independent transcription terminators were detected in the lactolisterin BU operon: the first in the intergenic region of the lliBU and abcT genes and the second at the end of operon. Deletion of the second transcription terminator did not influence production of the bacteriocin in lactococci.

Biological control of plant pathogens by Bacillus species.

Bacteria from the Bacillus group are microorganisms that inhabit a large number of different habitats. They are well known as producers of a wide array of antagonistic compounds of different structures, having between 5 to 8% of the total genome devoted to biosynthesis of secondary metabolites. Most important bioactive molecules from the genus Bacillus are non-ribosomally synthesized peptides and lipopeptides, polyketide compounds, bacteriocins and siderophores. Lipopeptides from Bacillus have very complex mechanisms of biosynthesis catalyzed by non-ribosomal peptide synthetases (NRPSs), large enzyme complexes with modular structure, with each module being in charge for the incorporation of a particular amino acid. In general, they have a broad spectrum of antagonistic activity against plant pathogenic bacteria, fungi and viruses. Most important molecules from this group, circular lipopeptides from surfactin, iturin and fengycin families affect the target cells on the membrane level. Bacillus strains exhibit their biocontrol capacity predominantly through inhibitory activity on the growth of plant pathogens, as well as inducing systemic resistance in plants and competing for ecological niches with plant pathogens. Our previous studies showed the presence of multiple biosynthetic operons for synthesis of non-ribosomal lipopeptides in the collection of natural isolates of Bacillus, with many strains having more than one of them. Several strains of Bacillus sp. that we have recently characterized showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed the presence of biosynthetic operons for iturin, bacillomycin, fengycin and surfactin in tested strains. Measurement of the kinetics of production of antimicrobial substances showed that, in most cases, synthesis started at the beginning of exponential phase of growth, reaching the maximum of antimicrobial activity at the beginning of the stationary growth phase and stayed at this level for the whole duration of observed period. Preparations of cell-free supernatants of tested strains were active against many fungal and bacterial pathogens, in vitro and in vivo. Mass spectrometry and HPTLC bioautography analysis of purified compounds confirmed the presence of lipopeptides of mentioned families, hence confirming the biocontrol capacity of Bacillus isolates.

Lactolisterin BU, a Novel Class II Broad-Spectrum Bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4.

Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti, a bacteriocin with analogy to aureocin A.IMPORTANCE Lactolisterin BU, a broad-spectrum leaderless bacteriocin produced by L. lactis subsp. lactis bv. diacetylactis BGBU1-4, expresses strong antimicrobial activity against food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU showed the highest similarity to aureocin-like bacteriocins produced by different bacteria. The operon for synthesis is located on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4, indicating possible horizontal transfer among producers.

Temperature, pH and Trimethoprim-Sulfamethoxazole Are Potent Inhibitors of Biofilm Formation by Stenotrophomonas maltophilia Clinical Isolates.

Stenotrophomonas maltophilia, an opportunistic pathogen usually connected with healthcare-associated infections, is an environmental bacterium. Intrinsic resistance to multiple antibiotics, with different virulence determinants in the last decade classified this bacterium in the group of global multiple drug resistant (MDR) organism. S. maltophilia clinical isolates, were collected from tertiary care pediatric hospital in Belgrade, Serbia to investigate influence of different factors on biofilm formation, kinetics of biofilm formation for strong biofilm producers and effect of trimethoprim-sulfamethoxazole (TMP/SMX) on formed biofilm. Most of the isolates (89.8%) were able to form a biofilm. Analysis of biofilm formation in different growth conditions showed that changing of temeperature and pH had the stronggest effect on biofilm formation almost equally in group of cystic fibrosis (CF) and non-CF strains. TMP/SMX in concentration of 50 µg/ml reduced completely 24 h old biofilms while concentration of 25 µg/ml effects formed biofilms in a strain dependent manner. Among strains able to form strong biofilm CF isolates formed biofilm slower than non-CF isolates, while shaking conditions did not affect biofilm formation. Swimming motility was detected in both CF and non-CF isolates, however more motile strain formed stronger biofilms. This study suggests that temperature, pH and TMP/SMX had the strongest influence on biofilm formation in analyzed collection of S. maltophilia. A positive correlation between motility and strength of formed biofilm was demonstrated.

Licheniocin 50.2 and Bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 Inhibit Biofilms of Coagulase Negative Staphylococci and Listeria monocytogenes Clinical Isolates.

BACKGROUND: Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates. METHODS: The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates. RESULTS: Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200-400 AU/ml for licheniocin 50.2 and 400-3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001). CONCLUSIONS: This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes.

Genotypic and Phenotypic Characterization of Stenotrophomonas maltophilia Strains from a Pediatric Tertiary Care Hospital in Serbia.

BACKGROUND: Stenotrophomonas maltophilia is an environmental bacterium and an opportunistic pathogen usually associated with healthcare-associated infections, which has recently been recognized as a globally multi-drug resistant organism. The aim of this study was genotyping and physiological characterization of Stenotrophomonas maltophilia isolated in a large, tertiary care pediatric hospital in Belgrade, Serbia, hosting the national reference cystic fibrosis (CF) center for pediatric and adult patients. METHODS: We characterized 42 strains of cystic fibrosis (CF) and 46 strains of non-cystic fibrosis (non-CF) origin isolated from 2013 to 2015 in order to investigate their genetic relatedness and phenotypic traits. Genotyping was performed using sequencing of 16S rRNA gene, Pulse Field Gel Electrophoresis (PFGE) and Multi locus sequencing typing (MLST) analysis. Sensitivity to five relevant antimicrobial agents was determined, namely trimethoprim/sulfamethoxazole (TMP/SMX), chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Surface characteristics, motility, biofilm formation and adhesion to mucin were tested in all strains. Statistical approach was used to determine correlations between obtained results. RESULTS: Most of the isolates were not genetically related. Six new sequence types were determined. Strains were uniformly sensitive to all tested antimicrobial agents. The majority of isolates (89.8%) were able to form biofilm with almost equal representation in both CF and non-CF strains. Swimming motility was observed in all strains, while none of them exhibited swarming motility. Among strains able to adhere to mucin, no differences between CF and non-CF isolates were observed. CONCLUSIONS: High genetic diversity among isolates implies the absence of clonal spread within the hospital. Positive correlation between motility, biofilm formation and adhesion to mucin was demonstrated. Biofilm formation and motility were more pronounced among non-CF than CF isolates.

Novel E. coli ST5123 Containing blaNDM-1 Carried by IncF Plasmid Isolated from a Pediatric Patient in Serbia.

New Delhi metallo-ß-lactamase (NDM) is a serious challenge to the treatment of infections and public health. Serbia has been designated as an endemic region for isolates carrying the blaNDM-1 gene, as well as one of several commonly proposed countries of origin. This is the first report of NDM-1-positive Escherichia coli from Serbia. A carbapenem-resistant clinical isolate of E. coli strain IMD989, isolated from the blood culture of a pediatric patient with leukemia, was subjected to antimicrobial susceptibility tests, molecular typing, and conjugation experiments. The strain exhibited resistance to meropenem and was classified as a novel sequence type, ST5123, belonging to E. coli phylogenetic group A. ST5123 showed similarity to veterinary isolates ST93 and ST3977. The blaNDM-1 gene was detected by polymerase chain reaction (PCR) and sequencing. Cloning and sequencing of genomic clones confirmed that strain IMD989 produces an NDM-1 variant. Conjugation experiments, pulsed-field gel electrophoresis, and Southern blot hybridization revealed that blaNDM-1 was located in IMD989 on a transmissible 80 kb plasmid, designated as pIMD989. PCR analysis confirmed that pIMD989 belongs to the IncF plasmid family. Propagation of IMD989 and selected transconjugants carrying pIMD989 over 14 days in solid media with and without antibiotic selection showed that pIMD989 is a stable plasmid.

Expression of bacteriocin LsbB is dependent on a transcription terminator.

The production of LsbB, leaderless class II bacteriocin, is encoded by genes (lsbB and lmrB) located on plasmid pMN5 in Lactococcus lactis BGMN1-5. Heterologous expression of the lsbB gene using the pAZIL vector (pAZIL-lsbB) in L. lactis subsp. cremoris MG7284 resulted in a significant reduction (more than 30 times) of bacteriocin LsbB expression. Subcloning and deletion experiments with plasmid pMN5 revealed that full expression of LsbB requires the presence of a complete transcription terminator located downstream of the lsbB gene. RNA stability analysis revealed that the presence of a transcription terminator increased the RNA stability by three times and the expression of LsbB by 30 times. The study of the influence of transcription terminator on the expression of other bacteriocin genes (lcnB, for lactococcin B production) indicated that this translational terminator likely functions in a lsbB-specific manner rather than in a general manner.

Isolation and Characterisation of Bacteriocinand Aggregation-Promoting Factor Production inLactococcus lactis ssp. lactis BGBM50 Strain.

Lactococcus lactis ssp. lactis BGBM50, a producer of lactococcin G and aggregation-promoting factor, was isolated from selected lactic acid bacteria taken from semi-hard cheese traditionally produced in the village Zanjic, Montenegro. Strain BGBM50 harbours a number of plasmids of different sizes. Plasmid curing experiments showed that genes for bacteriocin production are located on pBM140, a plasmid 140 kb in length. PCR analysis with primers specific for lactococcin Q and G genes gave fragment of the expected size. In addition, after plasmid curing of strain BGBM50, different derivatives with altered phenotypes were obtained, among them BGBM50-34 strain, which retained bacteriocin synthesis but had enhanced aggregation ability.

A Zn-dependent metallopeptidase is responsible for sensitivity to LsbB, a class II leaderless bacteriocin of Lactococcus lactis subsp. lactis BGMN1-5.

Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors.

Evaluation of lactic acid bacteria and yeast diversity in traditional white pickled and fresh soft cheeses from the mountain regions of Serbia and lowland regions of Croatia.

The goal of this study was the characterisation of indigenous lactic acid bacteria (LAB) and yeasts isolated from nine white pickled (BG) and nine fresh soft (ZG) artisanal cheeses collected in Serbia and Croatia. While LAB were present in all of the cheeses collected, yeasts were found in all BG cheeses but only in three ZG cheese samples. High LAB and yeast species diversity was determined (average H'(L)=0.4 and H'(Y)=0.8, respectively). The predominant LAB species in white pickled (BG) cheeses were Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides, while in fresh soft (ZG) cheeses the most dominant LAB species were L. lactis, Enterococcus faecalis, and Leuconostoc pseudomesenteroides. Among the 20 yeast species found, Debaryomyces hansenii, Candida zeylanoides, and Torulaspora delbrueckii were found to be predominant in BG cheeses, while Yarrowia lipolytica was predominant in ZG cheeses. The characterisation of metabolic and technological potentials revealed that 53.4% of LAB isolates produced antimicrobial compounds, 44.3% of LAB strains showed proteolytic activity, while most of the yeast species possessed either lipolytic or proteolytic activity. In conclusion, the results obtained in this study showed that the composition of LAB and yeast populations in white pickled and fresh soft cheeses is region specific. The knowledge gained in this study could eventually be used to select region specific LAB and yeast strains for the production of white pickled and fresh soft artisanal cheeses with geographically specific origins under controlled conditions.

Technological and probiotic potential of BGRA43 a natural isolate of Lactobacillus helveticus.

Lactobacillus helveticus BGRA43 is a human intestinal isolate showing antimicrobial activity, amongst others, against Yersinia enterocolitica, Shigella sonnei, Shigella flexneri, and Streptococcus pneumoniae. BGRA43 produces PrtH proteinase with proteolytic activity on both casein and ß-lactoglobulin (BLG). BGRA43 is able to reduce the allergenicity of BLG. Bioactive peptides released in BGRA43 fermented milk are potent modulators of innate immunity by modulating the production of proinflammatory cytokines IL-6 and TNF-α. BGRA43 is able to survive in simulated gastric and intestinal conditions. The growth of BGRA43 in milk results in a fast acidification lowering the milk pH to 4.53 generating mild, homogeneous, and viscous yogurt-like product. The strain BGRA43 grows suitably in pure cow or goat's milk as well as in milk containing inulin or nutrim even when they are used as the sole carbon source. It is suggested that strain BGRA43 could be used as a single-strain culture for the preparation of yogurt-like products from bovine or caprine milk. Overall, L. helveticus BGRA43 could be considered as a potential probiotic candidate with appropriate technological properties attractive for the dairy industry.

Prevalence of quinolone resistance and mutations in the topoisomerase genes in Salmonella enterica serotype Enteritidis isolates from Serbia.

The prevalence of quinolone resistance was studied in Salmonella enterica serotype Enteritidis isolates collected during 2005-2010 in Southern Backa County, Serbia. A total of 878 clinical isolates were examined, among which 19 (2.2%) nalidixic acid (NAL)-resistant S. Enteritidis were detected by selection on agar plates containing 64 mg/L NAL. Antimicrobial susceptibility of the isolates was tested by the agar dilution method. According to Clinical and Laboratory Standards Institute (CLSI) breakpoints, ciprofloxacin (CIP) resistance was not present in the strains. Multiple drug resistance was rare, and resistance to NAL was most often present as a single resistance property. All but one NAL-resistant S. Enteritidis showed reduced susceptibility to CIP [i.e. minimum inhibitory concentration (MIC)≥0.125 mg/L]. This isolate of human origin had a CIP MIC of 0.064 mg/L and DNA sequencing revealed that it contained an Asp87Gly gyrA mutation. Most of the remaining isolates had MICs for NAL and CIP of 256 mg/L and 0.256 mg/L, respectively. Mutations in the Asp87 codon resulted in substitutions to Asn in most of the isolates, but Asp87Gly and Ser83Phe exchanges were also detected. No mutations were present in the gyrB, parC or parE genes. Although CIP resistance was absent, reduced susceptibility characterised by mutations in gyrA was apparent among S. Enteritidis isolates from Serbia.

Cloning and expression of a novel lactococcal aggregation factor from Lactococcus lactis subsp. lactis BGKP1.

BACKGROUND: Aggregation may play a main role in the adhesion of bacteria to the gastrointestinal epithelium and their colonization ability, as well as in probiotic effects through co-aggregation with intestinal pathogens and their subsequent removal. The aggregation phenomenon in lactococci is directly associated with the sex factor and lactose plasmid co-integration event or duplication of the cell wall spanning (CWS) domain of PrtP proteinase. RESULTS: Lactococcus lactis subsp. lactis BGKP1 was isolated from artisanal semi-hard homemade cheese and selected due to its strong auto-aggregation phenotype. Subsequently, non-aggregating derivative (Agg-) of BGKP1, designated as BGKP1-20, was isolated, too. Comparative analysis of cell surface proteins of BGKP1 and derivative BGKP1-20 revealed a protein of approximately 200 kDa only in the parental strain BGKP1. The gene involved in aggregation (aggL) was mapped on plasmid pKP1 (16.2 kb), cloned and expressed in homologous and heterologous lactococci and enterococci. This novel lactococcal aggregation protein was shown to be sufficient for cell aggregation in all tested hosts. In addition to the aggL gene, six more ORFs involved in replication (repB and repX), restriction and modification (hsdS), transposition (tnp) and possible interaction with mucin (mbpL) were also located on plasmid pKP1. CONCLUSION: AggL is a new protein belonging to the collagen-binding superfamily of proteins and is sufficient for cell aggregation in lactococci.