Xenorhabdus khoisanae SB10 produces Lys-rich PAX lipopeptides and a Xenocoumacin in its antimicrobial complex.

BACKGROUND: Xenorhabdus spp. live in close symbiosis with nematodes of the Steinernema genus. Steinernema nematodes infect an insect larva and release their symbionts into the haemocoel of the insect. Once released into the haemocoel, the bacteria produce bioactive compounds to create a semi-exclusive environment by inhibiting the growth of bacteria, yeasts and molds. The antimicrobial compounds thus far identified are xenocoumacins, xenortides, xenorhabdins, indole derivatives, xenoamicins, bicornutin and a number of antimicrobial peptides. The latter may be linear peptides such as the bacteriocins xenocin and xenorhabdicin, rhabdopeptides and cabanillasin, or cyclic, such as PAX lipopeptides, taxlllaids, xenobactin and szentiamide. Thus far, production of antimicrobial compounds have been reported for Xenorhabdus nematophila, Xenorhabdus budapestensis, Xenorhabdus cabanillasii, Xenorhabdus kozodoii, Xenorhabdus szentirmaii, Xenorhabdus doucetiae, Xenorhabdus mauleonii, Xenorhabdus indica and Xenorhabdus bovienii. Here we describe, for the first time, PAX lipopeptides and xenocoumacin 2 produced by Xenorhabdus khoisanae. These compounds were identified using ultraperformance liquid chromatography, linked to high resolution electrospray ionisation mass spectrometry and tandem mass spectrometry. RESULTS: Cell-free supernatants of X. khoisanae SB10 were heat stable and active against Bacillus subtilis subsp. subtilis, Escherichia coli and Candida albicans. Five lysine-rich lipopeptides from the PAX group were identified in HPLC fractions, with PAX1' and PAX7 present in the highest concentrations. Three novel PAX7 peptides with putative enoyl modifications and two linear analogues of PAX1' were also detected. A small antibiotic compound, yellow in colour and λmax of 314 nm, was recovered from the HPLC fractions and identified as xenocoumacin 2. The PAX lipopeptides and xenocoumacin 2 correlated with the genes and gene clusters in the genome of X. khoisanae SB10. CONCLUSION: With UPLC-MS and MSe analyses of compounds in the antimicrobial complex of X. khoisanae SB10, a number of PAX peptides and a xenocoumacin were identified. The combination of pure PAX1' peptide with xenocoumacin 2 resulted in high antimicrobial activity. Many of the fractions did, however, contain labile compounds and some fractions were difficult to resolve. It is thus possible that strain SB10 may produce more antimicrobial compounds than reported here, as suggested by the APE Ec biosynthetic complex. Further research is required to develop these broad-spectrum antimicrobial compounds into drugs that may be used in the fight against microbial infections.

Differentiation between Bacillus amyloliquefaciens and Bacillus subtilis isolated from a South African sugarcane processing factory using ARDRA and rpoB gene sequencing.

A total of 104 exopolysaccharide (gum)-producing bacteria were isolated from the juice screen and juice tank in a sugarcane processing factory at times of low- and high dextran concentrations in the produced sugar. Dextran is an indicator of cane deterioration and sucrose loss after harvesting of the cane. The isolates were identified as Bacillus amyloliquefaciens (96 isolates) and Bacillus subtilis (eight isolates) based on restriction enzyme banding patterns of amplified 16S rRNA genes and rpoB gene sequence analysis. Exopolysaccharide production in sugarcane is normally associated with dextran produced by Leuconostoc mesenteroides. B. amyloliquefaciens, and to a lesser extent B. subtilis, could, however, also be responsible for exopolysaccharide (slime or gum) production in cane processing factories.

Polyacrylonitrile (PAN) nanofibres spun with copper nanoparticles: an anti-Escherichia coli membrane for water treatment.

Copper nanoparticles (CNPs) were mixed with polyacrylonitrile (PAN) and electrospun into nanofibres (CuPAN nanofibres). PAN nanofibres containing 1.0, 3.0 and 5.0% copper (w/v) displayed beads-on-string morphology with protrusions of copper particles. The diameter of the CuPAN nanofibres differed according to the copper content, ranging from 386 nm (1.0%, w/v, copper) to 922 nm (5.0%, w/v, copper). No chemical interaction of copper with PAN was observed when studied with X-ray diffraction, ATR-FTIR (attenuated total reflection-Fourier transform infrared) spectroscopy and TGA (thermogravimetric analysis). None of the CuPAN nanofibres showed signs of degradation after 7 days in water. Bacteria suspended in random mobility buffer and filtered through a 3% CuPAN nanofibre membrane (25 mm diameter, 75-80 µm thickness), at a filtration rate of 20 ml min-1, reduced the cell numbers of enterotoxigenic Escherichia coli (ETEC) from 3.3 × to 2.1 × 106 cfu ml-1 and methicillin-resistant Staphylococcus aureus (MRSA) from 1.2 × 10 to 1.3 × 103 cfu ml-1. Membranes produced with 1.0, 3.0 and 5.0% (w/v) CuPAN inhibited the growth of enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), ETEC and MRSA, as shown with LIVE/DEAD™ BacLight™ staining. Real-time bactericidal activity of CuPAN membranes was recorded by staining the cells with SYTO 9 and PI, followed by flow cytometry. Filter membranes made from CuPAN fibres may be used to reduce pathogenic E. coli cell numbers in potable water.

First Report of the Isolation of the Symbiotic Bacterium Photorhabdus luminescens subsp. laumondii Associated with Heterorhabditis safricana from South Africa.

Photorhabdus luminescens subsp. laumondii is closely associated with the entomopathogenic nematode Heterorhabditis bacteriophora and has, to date, not been isolated from other nematode species. This study is the first report of P. luminescens subsp. laumondii from two South African isolates of entomopathogenic nematodes, Heterorhabditis safricana SF281 and H. bacteriophora SF351. Both symbiotic bacterial strains are phenotypically closely related to P. luminescens subsp. laumondii previously isolated and described from H. bacteriophora. The genetic relatedness between P. luminescens subsp. laumondii strains SF281B and SF351B was confirmed by comparing 16S rDNA, recA, gyrB and gltX sequences with sequences of P. luminescens subsp. laumondii, including the type strain (TT01T) and strain E21.

First report of the symbiotic bacterium Xenorhabdus indica associated with the entomopathogenic nematode Steinernema yirgalemense.

The entomopathogenic nematode Steinernema yirgalemense is considered a promising agent in the biocontrol of insects. However, little is known about the bacteria living in symbiosis with the nematode. In this study, we have identified the only available bacterial strain (157-C) isolated from S. yirgalemense, as a member of the species Xenorhabdus indica. Identification was based on 16S rDNA, recA, dnaN, gltX, gyrB and infB gene sequence analyses. The relatedness of strain 157-C to the type strain of X. indica (DSM 17 382) was confirmed with DNA-DNA hybridization. The phenotypic characteristics of strain 157-C are similar to those described for the type strain of X. indica. This is the first report associating X. indica with S. yirgalemense.

Nisin F-loaded brushite bone cement prevented the growth of Staphylococcus aureus in vivo.

AIMS: To determine if nisin F-loaded self-setting brushite cement could control the growth of Staphylococcus aureus in vivo. METHODS AND RESULTS: Brushite cement was prepared by mixing equimolar concentrations of ß-tricalcium phosphate and monocalcium phosphate monohydrate. Nisin F was added at 5·0, 2·5 and 1·0% (w/w) and the cement moulded into cylinders. In vitro antibacterial activity was determined using a delayed agar diffusion assay. Release of nisin F from the cement was determined using BCA protein assays. Based on scanning electron microscopy and X-ray diffraction analysis, nisin F did not cause significant changes in cement structure or chemistry. Cement containing 5·0% (w/w) nisin F yielded the most promising in vitro results. Nisin F-loaded cement was implanted into a subcutaneous pocket on the back of mice and then infected with S. aureus Xen 36. Infection was monitored for 7 days, using an in vivo imaging system. Nisin F prevented S. aureus infection for 7 days and no viable cells were isolated from the implants. CONCLUSIONS: Nisin F-loaded brushite cement successfully prevented in vivo growth of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin F incorporated into bone cement may be used to control S. aureus infection in vivo.

Are fructophilic lactic acid bacteria (FLAB) beneficial to humans?

Fructophilic lactic acid bacteria (FLAB) are heterofermentative and related to the genera Fructilactobacillus, Convivina, Leuconostoc, Oenococcus and Weissella. Although they generally prefer fructose above glucose, obligate heterofermentative species will ferment glucose in the presence of external electron acceptors such as pyruvate and fructose. Little is known about the presence of FLAB in the human gut, let alone probiotic properties. In this review we discuss the possible role FLAB may have in the human gastro-intestinal tract (GIT) and highlight the advantages and disadvantages these bacteria may have in individuals with a diet high in fructose.

Influence of carbohydrates on the isolation of lactic acid bacteria.

AIMS: To determine the influence of carbohydrates on enrichment isolation of lactic acid bacteria from different niches. METHODS AND RESULTS: Lactic acid bacteria in three traditional fermented products in southern Africa (amasi, mahewu and tshwala) and in three fresh samples (two flowers and a fruit) were enrichment cultured in media supplemented with 13 different carbohydrates. Diversity of lactic acid bacteria was determined by PCR-denaturing-gradient gel electrophoresis. Carbohydrates used in enrichment media had a big impact on the isolation of lactic acid bacteria from fermented products. Depending on the carbohydrates tested, the number of species detected ranged from one to four in amasi, one to five in mahewu and one to three in tshwala. Fructose and mannitol selected for relatively higher numbers of lactic acid bacteria in fermented products. Specific relationships between substrates and lactic acid bacteria have been noted. On the other hand, small influences were found among carbohydrates tested in flowers and fruit. CONCLUSION: Carbohydrates have a big impact on the isolation of a variety of lactic acid bacteria in fermented food. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that reports the influence of carbohydrates on the enrichment of lactic acid bacteria.

Nisin F, intraperitoneally injected, may have a stabilizing effect on the bacterial population in the gastro-intestinal tract, as determined in a preliminary study with mice as model.

AIMS: To determine if nisin F has an effect on the bacterial population in the gastro-intestinal tract. METHODS AND RESULTS: Six male C57BL/6 mice were intraperitoneally injected with 200 µl sterile saline and six with nisin F (200 µl, equivalent to 640 arbitrary units). Fecal samples were collected before injection and 8, 24 and 48 h after injection, and the bacteria amplified by PCR-DGGE using 16S rDNA primers. The composition of the bacterial population in the gastro-intestinal tract (GIT) of mice that were injected with saline changed during 48 h, whereas the bacterial population in the GIT remained relatively unchanged in animals injected with nisin F. CONCLUSIONS: These results suggest that nisin F inhibits the growth of specific bacteria in the GIT within the first 4 h. Furthermore, the species remained repressed for at least 44 h after one intraperitoneal injection with nisin F. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report suggesting that nisin F may have a stabilizing effect on the bacterial population in the gastro-intestinal tract.

Nanofibers offer alternative ways to the treatment of skin infections.

Injury to the skin causes a breach in the protective layer surrounding the body. Many pathogens are resistant to antibiotics, rendering conventional treatment less effective. This led to the use of alternative antimicrobial compounds, such as silver ions, in skin treatment. In this review nanofibers, and the incorporation of natural antimicrobial compounds in these scaffolds, are discussed as an alternative way to control skin infections. Electrospinning as a technique to prepare nanofibers is discussed. The possibility of using these structures as drug delivery systems is investigated.

The ability of nisin F to control Staphylococcus aureus infection in the peritoneal cavity, as studied in mice.

AIMS: To determine the ability of nisin F to control systematic infection caused by Staphylococcus aureus, using C57BL/6 mice as a model. METHODS AND RESULTS: Twelve mice were intraperitoneally injected with 1 × 10(8) viable cells of Staph. aureus Xen 36 containing the modified Photorhabdus luminescence luxABCDE operon on plasmid pAUL-A Tn4001. After 4 h, six mice were intraperitoneally injected with 640 arbitrary units (AU) nisin F, and six were injected with sterile saline. Six mice, not infected with Staph. aureus, were treated with nisin F, and six not infected were left untreated. The viability of Staph. aureus Xen 36 was monitored over 48 h by recording photon emission levels. Nisin F suppressed Staph. aureus for 15 min in vivo. No abnormalities were recorded in blood analyses and internal organs of mice treated with nisin F. CONCLUSIONS: Nisin F suppressed the growth of Staph. aureus in the peritoneal cavity for at least 15 min. Re-emergence of Staph. aureus bioluminescence over the next 44 h suggests that nisin F was inactivated, most probably by proteolytic enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A single dosage of nisin F administered in the peritoneal cavity controlled the growth of Staph. aureus for at least 15 min in vivo.

Does the Future of Antibiotics Lie in Secondary Metabolites Produced by Xenorhabdus spp.? A Review.

The over-prescription of antibiotics for treatment of infections is primarily to blame for the increase in bacterial resistance. Added to the problem is the slow rate at which novel antibiotics are discovered and the many processes that need to be followed to classify antimicrobials safe for medical use. Xenorhabdus spp. of the family Enterobacteriaceae, mutualistically associated with entomopathogenic nematodes of the genus Steinernema, produce a variety of antibacterial peptides, including bacteriocins, depsipeptides, xenocoumacins and PAX (peptide antimicrobial-Xenorhabdus) peptides, plus additional secondary metabolites with antibacterial and antifungal activity. The secondary metabolites of some strains are active against protozoa and a few have anti-carcinogenic properties. It is thus not surprising that nematodes invaded by a single strain of a Xenorhabdus species are not infected by other microorganisms. In this review, the antimicrobial compounds produced by Xenorhabdus spp. are listed and the gene clusters involved in synthesis of these secondary metabolites are discussed. We also review growth conditions required for increased production of antimicrobial compounds.

Effect of a Multi-Species Probiotic on the Colonisation of Salmonella in Broilers.

Newly hatched broiler chickens are the most susceptible to Salmonella infections, especially during the first 24 h. At this age, the gut microbiome is not fully developed and offers little protection in the form of competitive exclusion. In this study, one group of newly hatched, Salmonella-free broilers were colonised with a multi-species probiotic (2.0 × 1010 to 8.9 × 1010 CFU per kg feed) for 28 days, consisting of Lactobacillus crispatus, Lactobacillus salivarius, Lactobacillus gallinarum, Lactobacillus johnsonii, Enterococcus faecalis and Bacillus amyloliquefaciens. Broilers in another group received oxytetracycline (200 mg/kg feed), instead of the probiotic, for 28 days. On days 9 and 10, broilers in both groups were gavaged with 9 × 107 CFU Salmonella Enteritidis A9, a pathogenic strain isolated from infected broilers. On day 14, Salmonella was detected in the ceca of 95% of broilers treated with the multi-species probiotic, but 2 weeks later, almost half of the birds (45%) had no Salmonella in their ceca. Similar results were recorded after 28 days of treatment with oxytetracycline. Only 10% of Salmonella-infected birds not treated were Salmonella-free on day 28. Growth performance, immune organ weight (spleen and bursa of Fabricius) and whole blood cell counts of birds treated with the multi-species probiotic and oxytetracycline, respectively, were similar to untreated and uninfected birds throughout the 28-day trial (p > 0.05). On day 14, serum lysozyme levels of broilers exposed to the probiotic were lower (8.0 µg/mL) compared with those of broilers treated with oxytetracycline (11.0 µg/mL). Although the multi-species probiotic and oxytetracycline stimulated the immune system, probiotics are safer to use than antibiotics and should be the preferred choice of treatment.

Lactobacillus and Bifidobacterium diversity in horse feces, revealed by PCR-DGGE.

Lactobacillus equi, Lactobacillus hayakitensis, Lactobacillus johnsonii, and Weissella confusa/cibaria were the dominant species in 12 South African horses. The Bifidobacterium-group was detected in the feces of only one of the 12 horses. Sequencing of the nested-PCR amplicon identified the Bifidobacterium-group as Parascardovia denticolens. Cell numbers of L. equi, L. hayakitensis, and W. confusa/cibaria were consistent in all samples. P. denticolens, Bifidodobacterium pseudolongum, and a phylogenetic relative of Alloscardovia omnicolens were rarely detected. L. equigenerosi, a dominant species in Japanese horses, was detected in the fecal samples of only one horse.

Nisin F in the treatment of respiratory tract infections caused by Staphylococcus aureus.

AIMS: To determine the antimicrobial activity of nisin F against Staphylococcus aureus in the respiratory tract. METHODS AND RESULTS: The respiratory tract of nonimmunosuppressed and immunosuppressed Wistar rats were colonized with 4 x 10(5) viable cells of S. aureus K and then treated by administering 8192 arbitrary units (AU) nisin F intranasal. Symptoms of pneumonia were detected in the trachea and lungs of immunosuppressed rats that had not been treated with nisin F. The trachea and lungs of immunosuppressed rats treated with nisin F were healthy. No significant differences were recorded in blood cell indices. The antimicrobial activity of low concentrations nisin F (80-320 AU ml(-1)) was slightly stimulated by lysozyme and lactoferrin. CONCLUSIONS: Nisin F inhibited the growth of S. aureus K in the respiratory tract of immunocompromised rats. Treatment with nisin F at 8192 AU proofed safe, as the trachea, lungs, bronchi and haematology of the rats appeared normal. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin F is nontoxic and may be used to control respiratory tract infections caused by S. aureus. This is, however, a preliminary study with an animal model and need to be confirmed with studies on humans.

Sodium acetate enhances hydrogen peroxide production in Weissella cibaria.

AIMS: To investigate hydrogen peroxide production by lactic acid bacteria (LAB) and to determine the key factors involved. METHODS AND RESULTS: Six strains of Weissella cibaria produced large amounts (2.2-3.2 mmol l(-1)) of hydrogen peroxide in GYP broth supplemented with sodium acetate, but very low accumulations in glucose yeast peptone broth without sodium acetate. Increased production of hydrogen peroxide was also recorded when strains of W. cibaria were cultured in the presence of potassium acetate, sodium isocitrate and sodium citrate. Oxidases and peroxidases were not detected, or were present at low levels in W. cibaria. However, strong nicotinamide adenine dinucleotide (NADH) oxidase activity was recorded, suggesting that the enzyme plays a key role in production of hydrogen peroxide by W. cibaria. CONCLUSIONS: Weissella cibaria produces large quantities of hydrogen peroxide in aerated cultures, in a process that is dependent on the presence of acetate in the culture medium. NADH oxidase is likely the key enzyme in this process. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study showing that sodium acetate, normally present in culture media of LAB, is a key factor for hydrogen peroxide production by W. cibaria. The exact mechanisms involved are not known.

Bacteriocin production and adhesion properties as mechanisms for the anti-listerial activity of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA.

Probiotics play an important role in maintaining a healthy and stable intestinal microbiota, primarily by preventing infection. Probiotic lactic acid bacteria (LAB) are known to be inhibitory to many bacterial enteric pathogens, including antibiotic-resistant strains. Whilst the positive role that probiotics have on human physiology, specifically in the treatment or prevention of specific infectious diseases of the gastro-intestinal tract (GIT) is known, the precise mechanistic basis of these effects remains a major research goal. In this study, molecular evidence to underpin the protective and anti-listerial effect of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA against orally administered Listeria monocytogenes EGDe in the GIT of mice is provided. Bacteriocins plantaricin 423 and mundticin ST4SA, produced by L. plantarum 423 and E. mundtii ST4SA, respectively, inhibited the growth of L. monocytogenes in vitro and in vivo. Bacteriocin-negative mutants of L. plantarum 423 and E. mundtii ST4SA failed to exclude L. monocytogenes EGDe from the gastrointestinal tract (GIT) of mice. Furthermore, L. plantarum 423 and E. mundtii ST4SA failed to inhibit recombinant strains of L. monocytogenes EGDe in vivo that expressed the immunity proteins of the two bacteriocins. These results confirmed that bacteriocins plantaricin 423 and mundticin ST4SA acted as anti-infective mediators in vivo. Compared to wild type strains, mutants of L. plantarum 423 and E. mundtii ST4SA, in which the adhesion genes were knocked out, were less effective in the exclusion of L. monocytogenes EGDe from the GIT of mice. This work demonstrates the importance of bacteriocin and adhesion genes as probiotic anti-infective mechanisms.

Characterization of the structural gene encoding nisin F, a new lantibiotic produced by a Lactococcus lactis subsp. lactis isolate from freshwater catfish (Clarias gariepinus).

Lactococcus lactis F10, isolated from freshwater catfish, produces a bacteriocin (BacF) active against Staphylococcus aureus, Staphylococcus carnosus, Lactobacillus curvatus, Lactobacillus plantarum, and Lactobacillus reuteri. The operon encoding BacF is located on a plasmid. Sequencing of the structural gene revealed no homology to other nisin genes. Nisin F is described.

A class IIa peptide from Enterococcus mundtii inhibits bacteria associated with otitis media.

Peptide ST4SA, produced by Enterococcus mundtii ST4SA, inhibits the growth of Acinetobacter baumannii, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Streptococcus pneumoniae and Gram-positive bacteria isolated from patients diagnosed with middle ear infections. The peptide adsorbed at a level of 94% to S. pneumoniae 40, Pseudomonas aeruginosa 25 and E. faecium HKLHS. Low concentrations of peptide ST4SA (51200 arbitrary units (AU)/mL) caused DNA and enzyme leakage from target cells, whilst 1638400AU/mL caused cell lysis. No decrease in antimicrobial activity was observed when tested on solid medium with human blood as base. Peptide ST4SA revealed a similar level of activity compared with tetracycline (30 microg), but much higher activity compared with nasal sprays, aminoglycosides, cephalosporins, fluoroquinolones, lincosamides, macrolides, nitroimidazole, penicillin, quinolones, sulphonamides, chloramphenicol, furazolidone, fusidic acid, rifampicin, trimethoprim, trimethoprim/sulfamethoxazole and vancomycin when tested in vitro. Peptide ST4SA dissipates the proton-motive force and may be used in the treatment of multidrug-resistant strains where antibiotics are excluded from cells by efflux pumps dependent on the membrane proton gradient.

Effect of gastro-intestinal conditions on the growth of Enterococcus mundtii ST4SA, and production of bacteriocin ST4SA recorded by real-time PCR.

Enterococcus mundtii ST4SA, isolated from soybeans, produces a 3950 Da bacteriocin (bacST4SA) active against a variety of Gram-positive and Gram-negative bacteria, including human pathogens. In this study, the effect of gastro-intestinal conditions on the survival of strain ST4SA and production of bacST4SA was studied. Strain ST4SA was cultured in MRS broth at different pH and in MRS broth supplemented with bile, pancreatic enzymes, and contents of the stomach and small intestine of pigs, respectively. After 12 and 24 h at 37 degrees C, cells were harvested, RNA isolated and cDNA prepared. Expression of the genes encoding bacST4SA, RecA, GroES and 23 S rRNA was studied by real-time PCR (RT-PCR). No significant up- or down-regulation of the genes were recorded, except when cells were grown in MRS at pH 3.5. In this case only RecA and GroES were up-regulated. Growth of strain ST4SA and production of bacST4SA are not affected by conditions in the lower intestine and the strain could be used as a probiotic.