Aims: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. Methods and Results: Random isolates (n ¼ 509) were collected from breast milk samples (n ¼ 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7AE5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12AE5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. Conclusions: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. Significance and Impact of the Study: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.

BackgroundThe implementation of novel chassis organisms to be used as microbial cell factories in industrial applications is an intensive research field. Lactococcus lactis, which is one of the most extensively studied model organisms, exhibits superior ability to be used as engineered host for fermentation of desirable products. However, few studies have reported about genome reduction of L. lactis as a clean background for functional genomic studies and a model chassis for desirable product fermentation.ResultsFour large nonessential DNA regions accounting for 2.83% in L. lactis NZ9000 (L. lactis 9 k) genome (2,530,294 bp) were deleted using the Cre-loxP deletion system as the first steps toward a minimized genome in this study. The mutants were compared with the parental strain in several physiological traits and evaluated as microbial cell factories for heterologous protein production (intracellular and secretory expression) with the red fluorescent protein (RFP) and the bacteriocin leucocin C (LecC) as reporters. The four mutants grew faster, yielded enhanced biomass, achieved increased adenosine triphosphate content, and diminished maintenance demands compared with the wild strain in the two media tested. In particular, L. lactis 9 k-4 with the largest deletion was identified as the optimum candidate host for recombinant protein production. With nisin induction, not only the transcriptional efficiency but also the production levels of the expressed reporters were approximately three- to fourfold improved compared with the wild strain. The expression of lecC gene controlled with strong constitutive promoters P5 and P8 in L. lactis 9 k-4 was also improved significantly.ConclusionsThe genome-streamlined L. lactis 9 k-4 outcompeted the parental strain in several physiological traits assessed. Moreover, L. lactis 9 k-4 exhibited good properties as platform organism for protein production. In future works, the genome of L. lactis will be maximally reduced by using our specific design to provide an even more clean background for functional genomics studies than L. lactis 9 k-4 constructed in this study. Furthermore, an improved background will be potentially available for use in biotechology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0616-2) contains supplementary material, which is available to authorized users.

Nisin produced by Lactococcus lactis subsp. lactis is a 34-residue antibacterial polypeptide and belongs to a group of post-translationally modified peptides, lantibiotics, with dehydrated residues and cyclic amino acids, lanthionines. These modifications are supposed to be made by enzymes encoded by lanB and lanC genes, found only in biosynthetic operons encoding lantibiotics. To analyse the extent of modification, His-tagged nisin precursors were expressed in nisB and nisC mutant strains. The His-tagged nisin precursors were purified from the cytoplasm of the cells, as lack of NisB or NisC activity impaired translocation of the nisin precursor. The purified His-tagged polypeptides were analysed with trypsin digestion followed by nisin bioassay, SDS-PAGE, Nterminal sequencing and mass spectroscopy. According to the results, nisin precursors from the strain lacking NisB activity were totally unmodified, whereas nisin precursors from the strain lacking NisC activity, but having NisB activity, were dehydrated and devoid of normal lanthionine formation. This is the first experimental evidence showing that NisB is required for dehydration and NisC for correct lanthionine formation in nisin maturation.

The lantibiotic nisin is produced by several strains of Lactococcus lactis subsp. lactis. The chromosomally located gene cluster nisABTC/PRKf€G is required for biosynthesis, development of immunity, and regulation of gene expression. Inframe deletions in the nisB and nisT genes, and disruption of nisC by plasmid integration, eliminated nisin production and resulted in a strongly reduced level of immunity of the strains. The transcription of two nisin operons was inactivated in these mutant strains, but could be restored by addition of small amounts of nisin to growing cultures. The immunity levels of the mutants were also raised by adding nisin to growing cultures, albeit not to wild-type level. A strain with an in-frame deletion in the nisl gene was still able to produce active nisin, but the production and immunity levels were markedly lower. By measuring immunity levels of the knock-out strains and determining mRNA levels, it is concluded that Nisl has an important function for nisin immunity and must cooperate with nisf€G-encoded proteins to provide a high level of immunity. Maximal immunity could not be obtained in the mutant strains, probably because the wild-type transcription levels from nisA and nisf promoters are not reached when essential nis genes are disrupted. Using Southern hybridization with a consensus promoter probe, no other DNA sequences similar to the nisA and nisf promoters could be detected, indicating that these two elements are probably the only ones in the chromosome regulated by nisin and are thus the only ones involved in the regulation of producer immunity.

Nisin is a small post-translationally modified lanthionine-containing peptide (lantibiotic) produced by certain Lactococcus lactis strains which has a high antimicrobial activity against several pathogenic Gram-positive bacteria. Northern blots and RT/PCR analyses of the nisin-producing strain N8 revealed that the nisZBTC/PRKF€G gene cluster, responsible for nisin biosynthesis, immunity and regulation, consists of two operons, nisZBTC/PRK and nisFEG. The promoter of the nisF€G operon was mapped. The -35 to -1 region upstream of the transcription start of the nisF€G promoter showed 73% identity with the corresponding region upstream of the nisA and nisZ gene. In contrast to earlier reports, nisin was found to be secreted during the early stages of growth as well as later in the growth cycle. The secreted nisin was adsorbed on the surface of the cells and was released to the medium during mid-exponential growth, when the pH in the medium fell below 55. In nisZB antisense and nisT deletion mutant strains constructed in this study the transcription of the nisin operons, nisin production and immunity were lost. Provision of external nisin restored the transcription of both operons in the mutant strains, showing that the operons are coordinately regulated by mature nisin. Nisin induction of the mutant strains also resulted in an increased amount of the Nisl protein and an increase in the level of immunity. Induction using higher concentrations of nisin yielded a higher level of immunity. These results showed that the nisin promoters are under positive control in an autoregulatory manner and that antimicrobial peptides can also function as signal molecules.

A gene locus of Bacillus subtilis identified by mutations (prs) conferring a defect in protein secretion was cloned from a lambdaGEM-11 expression library. The sites of three closely linked prs mutations (prs-3, prs-29 and prs-40) were found to reside in a 5.3 kb DNA fragment, which also complemented the secretion defect in prs-3 and prs-29 mutants. Partial sequencing of the fragment showed that these three mutations affect one distinct gene (prsA) encoding a putative protein of 292 amino acids (33 kDa). Sequence analysis indicated the PrsA protein to be a lipoprotein located outside the cytoplasmic membrane. Thirty percent identity was shown to the PrtM protein of Lactococcus lactis, which is involved in the maturation of an exported proteinase. The phenotypes of prsA mutants and the structural similarity of PrsA with PrtM suggest that PrsA may have a novel function at a late phase in protein export.

Characterization by partial 16S rRNA gene sequencing, ribotyping, and green fluorescent protein-based nisin bioassay revealed that 6 of 20 human milk samples contained nisin-producing Lactococcus lactis bacteria. This suggests that the history of humans consuming nisin is older than the tradition of consuming fermented milk products.One of the most studied bacteriocins, nisin is naturally produced by Lactococcus lactis. Both nisin variants A and Z, with a difference of an amino acid (1), are approved for use in foodstuffs by food additive legislating bodies in the United States (Food and Drug Administration), the United Kingdom, and the European Union (16). Nisin is employed in the dairy industry to inhibit Clostridium botulinum and Bacillus cereus, due to its capacity to prevent spore germination (7,20). Nisin was first identified in fermented cow milk (13). Since then, it has been isolated from various milk and dairy products (8, 16) as well as from plant material (5, 16) and river water (21). In this study, human milk was screened for bacteria to reveal antibacterial activity caused by nisin producers.Early lactational (within 80 days of birth) human milk samples (n ϭ 20) were collected in southern Finland from healthy first-time deliverers and from mothers with several children. Two milk samples were received within a month from one donor. The donors were requested to collect milk in sterile test tubes with minimal skin contact. The fresh milk was screened within hours of delivery for bacteria with antibacterial properties by the agar diffusion test (17). Samples were spotted on nonselective Luria-Bertani agar (14) overlaid with 100 l (at an optical density at 600 nm of 1.6) of Micrococcus luteus A1 NCIMB86166 (National Collection of Industrial and Marine Bacteria), a strain sensitive to many antibacterial substances. Plates covered with M. luteus were allowed to dry with the lid open before 30-l milk sampling and were incubated overnight at 37°C in an aerobic atmosphere. Seven samples from six different mothers contained strains with strong antibacterial activity. Of the 38 isolated strains, 20 colonies produced a clear inhibition zone around M. luteus and were isolated on plates of M17 agar (Oxoid Ltd., Basingstoke, England) with 0.5% (wt/ vol) glucose (M17G) and grown overnight at 30°C.The 20 isolated inhibitory strains from human milk were characterized by partial 16S rRNA gene sequencing. A region of the 16S rRNA gene was amplified by 29 cycles of PCR (consisting of 30 s at 94°C, 60 s at 55°C, and 90 s at 72°C, with a final 120-s extension step at 72°C) with purified chromosomal DNA (18) from the strains as template and using universal primers pA (5Ј AGA GTT TGA TCC TGG CTC AG 3Ј) and pEЈ (5Ј CCG TCA ATT CCT TTG AGT TT 3Ј) (3). The amplified 900-bp fragments were harvested from low-meltingpoint gel LM-3 (Pronadisa, Madrid, Spain), purified with chloroform-propanol (14) The seven L. lactis strains (LL3, 310, 2410, 3A, 4B, 6A, and 6B) were further characterized by the RiboPrinter and compared to the ribop...

Parkinson’s disease (PD) is the most prevalent movement disorder known and predominantly affects the elderly. It is a progressive neurodegenerative disease wherein α-synuclein, a neuronal protein, aggregates to form toxic structures in nerve cells. The cause of Parkinson’s disease (PD) remains unknown. Intestinal dysfunction and changes in the gut microbiota, common symptoms of PD, are evidently linked to the pathogenesis of PD. Although a multitude of studies have investigated microbial etiologies of PD, the microbial role in disease progression remains unclear. Here, we show that Gram-negative sulfate-reducing bacteria of the genus Desulfovibrio may play a potential role in the development of PD. Conventional and quantitative real-time PCR analysis of feces from twenty PD patients and twenty healthy controls revealed that all PD patients harbored Desulfovibrio bacteria in their gut microbiota and these bacteria were present at higher levels in PD patients than in healthy controls. Additionally, the concentration of Desulfovibrio species correlated with the severity of PD. Desulfovibrio bacteria produce hydrogen sulfide and lipopolysaccharide, and several strains synthesize magnetite, all of which likely induce the oligomerization and aggregation of α-synuclein protein. The substances originating from Desulfovibrio bacteria likely take part in pathogenesis of PD. These findings may open new avenues for the treatment of PD and the identification of people at risk for developing PD.