Impact of Environmental Factors on Bacteriocin Promoter Activity in Gut-Derived Lactobacillus salivarius.

Bacteriocin production is regarded as a desirable probiotic trait that aids in colonization and persistence in the gastrointestinal tract (GIT). Strains of Lactobacillus salivarius, a species associated with the GIT, are regarded as promising probiotic candidates and have a number of associated bacteriocins documented to date. These include multiple class IIb bacteriocins (salivaricin T, salivaricin P, and ABP-118) and the class IId bacteriocin bactofencin A, which show activity against medically important pathogens. However, the production of a bacteriocin in laboratory media does not ensure production under stressful environmental conditions, such as those encountered within the GIT. To allow this issue to be addressed, the promoter regions located upstream of the structural genes encoding the L. salivarius bacteriocins mentioned above were fused to a number of reporter proteins (green fluorescent protein [GFP], red fluorescent protein [RFP], and luciferase [Lux]). Of these, only transcriptional fusions to GFP generated signals of sufficient strength to enable the study of promoter activity in L. salivarius. While analysis of the class IIb bacteriocin promoter regions indicated relatively weak GFP expression, assessment of the promoter of the antistaphylococcal bacteriocin bactofencin A revealed a strong promoter that is most active in the absence of the antimicrobial peptide and is positively induced in the presence of mild environmental stresses, including simulated gastric fluid. Taken together, these data provide information on factors that influence bacteriocin production, which will assist in the development of strategies to optimize in vivo and in vitro production of these antimicrobials.

The Lantibiotic Lacticin 3147 Prevents Systemic Spread of Staphylococcus aureus in a Murine Infection Model.

The objective of this study was to investigate the in vivo activity of the lantibiotic lacticin 3147 against the luminescent Staphylococcus aureus strain Xen 29 using a murine model. Female BALB/c mice (7 weeks old, 17 g) were divided into groups (n = 5) and infected with the Xen 29 strain via the intraperitoneal route at a dose of 1 × 10(6) cfu/animal. After 1.5 hr, the animals were treated subcutaneously with doses of phosphate-buffered saline (PBS; negative control) or lacticin 3147. Luminescent imaging was carried 3 and 5 hours postinfection. Mice were then sacrificed, and the levels of S. aureus Xen 29 in the liver, spleen, and kidneys were quantified. Notably, photoluminescence and culture-based analysis both revealed that lacticin 3147 successfully controlled the systemic spread of S. aureus in mice thus indicating that lacticin 3147 has potential as a chemotherapeutic agent for in vivo applications.

Bioengineering of a Nisin A-producing Lactococcus lactis to create isogenic strains producing the natural variants Nisin F, Q and Z.

Nisin is the prototypical example of the lantibiotic family of antimicrobial peptides and has been employed as a food preservative for over half a century. It has also attracted attention due to its potency against a number of multidrug-resistant clinical pathogens. Nisin A is the originally isolated form of Nisin and a further five natural variants have been described which differ by up to 10 amino acids (of 34 in total in Nisin A). Nisins A, Z, F and Q are produced by Lactococcus lactis, while Nisins U and U2 are produced by Streptococcus sp. In this study we bioengineered the nisA gene of a Nisin A producer to generate genes encoding Nisins Z, F, Q, U and U2. We determined that while active Nisin Z, F and Q can be produced against this genetic background, active forms of Nisin U and U2 are not generated. Minimum inhibitory concentration studies with Nisin A, Z, F and Q variants against a series of different clinically significant pathogens establish differences in specific activities against selected targets. Nisin F was most impressive, being the most active, or one of the most active, against the MRSA strain ST 525, EC 676, EC 725, VISA 22900, VISA 22781, hVISA 35197, Staphylococcus aureus 8325-4 and L. lactis HP. Nisin Z was most active against ST 299, hVISA 32683 and, together with Nisin F, HP but had contrastingly poor activity against ST 525, EC 676 and 8325-4. Nisin F, Q and A exhibited similar potency against VISA 22900. This was the only target against which Nisin Q and Nisin A were among the most active variants.

A comparison of the activities of lacticin 3147 and nisin against drug-resistant Staphylococcus aureus and Enterococcus species.

OBJECTIVES: Our goal was to compare the activities of lacticin 3147 and nisin, two of the most well characterized lantibiotics, against antibiotic-resistant staphylococci and enterococci. METHODS: We determined the MICs of lacticin 3147 and nisin for 20 strains of methicillin-resistant Staphylococcus aureus (MRSA), 20 strains of vancomycin-resistant enterococci (VRE), 6 strains of S. aureus with intermediate resistance to vancomycin (VISA), 5 strains of heterogeneous vancomycin-intermediate S. aureus (hVISA) and 4 strains of S. aureus that are susceptible to methicillin. RESULTS: Lacticin 3147 displayed potent activity against VRE with MIC values between 1.9 and 7.7 mg/L, and varying levels of activity against S. aureus strains (MRSA, 1.9-15.4 mg/L; laboratory strains, >or=15.4 mg/L; hVISA, 15.4-30.9 mg/L; VISA, >or=61.8 mg/L). Nisin was more active against the S. aureus strains in general (MRSA and laboratory strains, 0.5-4.1 mg/L; VISA and hVISA, 2 to >or=8.3 mg/L), but was less effective than lacticin 3147 against VRE (2 to >or=8.3 mg/L). CONCLUSIONS: Nisin is more effective against S. aureus whereas lacticin 3147 possesses greater potency against VRE. The modifications responsible for the vancomycin-resistant phenotypes of hVISA and VISA strains also provide protection against the two lantibiotics.

Discovery of medically significant lantibiotics.

The emergence of drug-resistant pathogens such as staphylococci and enterococci in the hospital setting has long being recognized as a serious clinical problem. Staphylococcus aureus is the causative agent of many nosocomial infections from minor skin abscesses to serious, potentially life threatening diseases such as bone and soft tissue intra-surgical infections, sepsis and invasive endocarditis, while enterococci are responsible for nosocomial bacteraemia, surgical wound infections and endocarditis. The most infamous drug-resistant forms of these include MRSA (methicillin resistant S. aureus), VISA (vancomycin insensitive S. aureus), hVISA (heterogenous vancomycin insensitive S. aureus) and VRE (vancomycin resistant S. aureus). While enhanced hygiene awareness is essential to any solution, the identification of effective novel antimicrobial compounds remains a major goal in eradicating these and other infections caused by multi-drug resistant pathogens. In recent years a class of antimicrobial peptides, the Lantibiotics, have been the focus of an ever increasing level of attention. This interest has been prompted by an enhanced appreciation of the mode of action of these peptides (including, in many cases, the ability to bind lipid II) and their frequently high levels of antimicrobial activity. Here we review lantibiotic-related issues in drug discovery, outline the strategies that have been employed to identify these peptides and summarize the use of bioengineering to generate enhanced forms of these peptides as well as the application of the associated biological machinery to generate novel forms of existing pharmaceutical compounds. In so doing we highlight how some, or all, of these approaches have the potential to result in the development of clinically important drugs.

Isolation and analysis of bacteria with antimicrobial activities from the marine sponge Haliclona simulans collected from Irish waters.

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.