GS-2: A Novel Broad-Spectrum Agent for Environmental Microbial Control.

The environmental control of microbial pathogens currently relies on compounds that do not exert long-lasting activity on surfaces, are impaired by soil, and contribute to the growing problem of antimicrobial resistance. This study presents the scientific development and characterization of GS-2, a novel, water-soluble ammonium carboxylate salt of capric acid and L-arginine that demonstrates activity against a range of bacteria (particularly Gram-negative bacteria), fungi, and viruses. In real-world surface testing, GS-2 was more effective than a benzalkonium chloride disinfectant at reducing the bacterial load on common touch-point surfaces in a high-traffic building (average 1.6 vs. 32.6 CFUs recovered from surfaces 90 min after application, respectively). Toxicology testing in rats confirmed GS-2 ingredients were rapidly cleared and posed no toxicities to humans or animals. To enhance the time-kill against Gram-positive bacteria, GS-2 was compounded at a specific ratio with a naturally occurring monoterpenoid, thymol, to produce a water-based antimicrobial solution. This GS-2 with thymol formulation could generate a bactericidal effect after five minutes of exposure and a viricidal effect after 10 min of exposure. Further testing of the GS-2 and thymol combination on glass slides demonstrated that the compound retained bactericidal activity for up to 60 days. Based on these results, GS-2 and GS-2 with thymol represent a novel antimicrobial solution that may have significant utility in the long-term reduction of environmental microbial pathogens in a variety of settings.

Susceptibility of intracellular Coxiella burnetii to antimicrobial peptides in mouse fibroblast cells.

Q fever is a zoonotic disease caused by Coxiella burnetii, an obligate intracellular bacterium that resides inside a phagolysosome-like niche. Chronic Q fever is typified by endocarditis, and is treated with multiple antibiotics for at least 18 months. The discovery of clinical C. burnetii isolates resistant to the first-line antibiotic doxycycline, and the problematic nature of chronic Q fever treatment have demonstrated the need for improved treatment regimes. To search for alternative antimicrobial agents, we assessed the effect of 26 antimicrobial peptides (AMPs) on the intracellular growth of C. burnetii in L929 cells at a concentration of 25 µM or their maximal non-cytotoxic concentration. Among the peptides tested, A3-APO, Cath-BF, δ-Hemolysin, Octa-1, P5 and Pleurocidin were able to significantly reduce both the total bacterial cell number and the host cell bacterial burden (average bacterial number per host cell). Combining selected AMPs with Chariot, a non-covalent carrier peptide, did not increase treatment potency when non-cytotoxic concentrations were used, with the exception of P5, which remained active at a concentration of 1.6 µM (1.8 µg/mL). Combining AMPs with each other did not further improve AMP potency, with some treatment combinations increasing the growth rate of C. burnetii by >3-fold. This is the first description of AMP cellular penetration to exhibit inhibitory affect on intracellular C. burnetii growth. These results are the first step in the development of a non-traditional antibiotic treatment for Q fever.

Panning of a phage display library against a synthetic capsule for peptide ligands that bind to the native capsule of Bacillus anthracis.

Bacillus anthracis is the causative agent of anthrax with the ability to not only produce a tripartite toxin, but also an enveloping capsule comprised primarily of γ-D-glutamic acid residues. The purpose of this study was to isolate peptide ligands capable of binding to the native capsule of B. anthracis from a commercial phage display peptide library using a synthetic form of the capsule consisting of 12 γ-D-glutamic acid residues. Following four rounds of selection, 80 clones were selected randomly and analysed by DNA sequencing. Four clones, each containing a unique consensus sequence, were identified by sequence alignment analysis. Phage particles were prepared and their derived 12-mer peptides were also chemically synthesized and conjugated to BSA. Both the phage particles and free peptide-BSA conjugates were evaluated by ELISA for binding to encapsulated cells of B. anthracis as well as a B. anthracis capsule extract. All the phage particles tested except one were able to bind to both the encapsulated cells and the capsule extract. However, the peptide-BSA conjugates could only bind to the encapsulated cells. One of the peptide-BSA conjugates, with the sequence DSSRIPMQWHPQ (termed G1), was fluorescently labelled and its binding to the encapsulated cells was further confirmed by confocal microscopy. The results demonstrated that the synthetic capsule was effective in isolating phage-displayed peptides with binding affinity for the native capsule of B. anthracis.

Potent antimicrobial peptides with selectivity for Bacillus anthracis over human erythrocytes.

In this study, 39 antimicrobial peptides, most with documented low haemolytic activity and potent efficacy against Gram-negative and Gram-positive bacteria, were evaluated for their haemolytic activity against human red blood cells as well as their antimicrobial activity against Escherichia coli, Burkholderia thailandensis, Bacillus globigii and Bacillus anthracis. The majority of the peptides had a minimum inhibitory concentration (MIC) of <10 µM against B. globigii. However, only eight of these (CaLL, Ci-MAM-A24, LLaMA, Ltc2a, OV-5, papillosin, smapspin and smapspin-G) had a MIC<10 µM against B. anthracis. All except one (papillosin) were ineffective at 100 µM against B. thailandensis and none had potent activity against E. coli. Potent activity against B. anthracis was associated with significant haemolytic activity, but the ratio of the concentration of peptide that caused 50% haemolysis to the concentration that inhibited growth of B. anthracis by 50% (the therapeutic index) varied from 0.8 to 34.2. Two peptides (papillosin and Ltc2a) had a therapeutic index >30 and could be considered as candidates for further development for potential medical countermeasures against anthrax. Although B. globigii has often been used as a non-pathogenic simulant for B. anthracis, in this study it was found that the sensitivity of B. globigii to peptides was not a reliable predictor of the sensitivity of B. anthracis to the same peptides.

Analogues of peptide SMAP-29 with comparable antimicrobial potency and reduced cytotoxicity.

SMAP-29 (sheep myeloid antimicrobial peptide-29) is a peptide with potent antibacterial properties. However, it is also highly cytotoxic both to human red blood cells (hRBCs) and human embryonic kidney (HEK) cells. In this study, some of the amino acids of SMAP-29 were changed in an attempt to reduce haemolytic activity whilst maintaining high antibacterial efficacy. These analogues, plus other analogues described in the literature with potent antimicrobial activity against Gram-positive bacteria coupled with no or low haemolytic activity, were evaluated for their cytotoxicity (hRBCs and HEK cells) as well as antimicrobial efficacy against two Gram-positive (Bacillus anthracis and Bacillus globigii) and two Gram-negative bacteria (Escherichia coli and Burkholderia thailandensis). The analogues previously described in the literature were found to have low antibacterial and haemolytic activity. Two of the designed analogues had comparable antibacterial efficacy with SMAP-29 against B. anthracis but reduced haemolytic activity and therefore had a therapeutic index that was enhanced 2.3-2.6-fold over that of SMAP-29.

Isolation, kinetic analysis, and structural characterization of an antibody targeting the Bacillus anthracis major spore surface protein BclA.

One method of laboratory- or field-based testing for anthrax is detection of Bacillus anthracis spores by high-affinity, high specificity binding reagents. From a pool of monoclonal antibodies, we selected one such candidate (A4D11) with high affinity for tBclA, a truncated version of the B. anthracis exosporium protein BclA. Kinetic analysis utilising both standard and kinetic titration on a Biacore biosensor indicated antibody affinities in the 300 pM range for recombinant tBclA, and the A4D11 antibody was also re-formatted into scFv configuration with no loss of affinity. However, assays against B. anthracis and related Bacilli species showed limited binding of intact spores as well as significant cross-reactivity between species. These results were rationalized by determination of the three-dimensional crystallographic structure of the scFv-tBclA complex. A4D11 binds the side of the tBclA trimer, contacting a face of the antigen normally packed against adjacent trimers within the exosporium structure; this inter-spore interface is highly conserved between Bacilli species. Our results indicate the difficulty of generating a high-affinity antibody to differentiate between the highly conserved spore structures of closely related species, but suggest the possibility of future structure-based antibody design for this difficult target.

Characterisation and evaluation of synthetic antimicrobial peptides against Bacillus globigii, Bacillus anthracis and Burkholderia thailandensis.

Antimicrobial peptides (AMPs) are produced by all forms of living organisms and represent a novel class of antibiotics to treat infectious diseases. In this study, 29 AMPs of varying length and characteristics were synthesised chemically and were evaluated for their ability to inhibit the growth of Bacillus globigii, Bacillus anthracis and Burkholderia thailandensis. Amongst the peptides tested, sheep myeloid antimicrobial peptide-29 (SMAP-29) was the most potent, inhibiting both B. globigii and B. anthracis at submicromolar concentrations. However, SMAP-29 was less effective against B. thailandensis (minimum inhibitory concentration of 71 microM). Haemolytic activity and cytotoxicity were determined using human blood cells and human embryonic kidney 293S cells, respectively. Most of the peptides tested showed varying degrees of haemolytic activity and cytotoxicity, with SMAP-29 being highly haemolytic and cytotoxic under the conditions tested. Nevertheless, strategies to reduce toxicity whilst maintaining high antimicrobial activity are worth pursuing in light of the results obtained.

Surface plasmon resonance detection of ricin and horticultural ricin variants in environmental samples.

A rapid, sensitive and robust immunoassay based on a commercial surface plasmon resonance (SPR) instrument (Biacore X) was developed for the detection of ricin in environmental samples. A total of 10 monoclonal antibodies were evaluated for their ability to recognise both a commercial ricin and horticultural ricin variants extracted from six different cultivars of Ricinus communis. Two suitable antibodies (7G12 and TFTA) were identified because of their strong affinity to all six ricin variants. The antibody 7G12 was used as the capture ligand in the SPR system. The assay was linear over a wide range of ricin concentrations (up to at least 750 ng/ml) with a limit of detection of 0.5 ng/ml. The assay was highly reproducible (coefficient of variation was less than 5%), and was able to detect all six ricin variants and environmental samples.

Properties and applications of antimicrobial peptides in biodefense against biological warfare threat agents.

Recent advances in knowledge of the properties of antimicrobial peptides (AMPs) are reviewed. AMPs are typically small, positively charged, amphipathic peptides that interact electrostatically and non-stereospecifically with the bacterial cell membrane, resulting in its permeabilization and cell death. Classes of AMPs, their mechanisms of action, hemolytic activity, and cytotoxicity towards host cells are discussed. A particular focus is AMPs with potential for use in defense against biological warfare agents. Some AMPs cytotoxic to Bacillus anthracis have been described. Synthesis of these peptides in multivalent form leads to a synergistic increase in antibacterial activity. Strategies to enhance the potency, stability, and selectivity of AMPs are discussed.

Construction, crystal structure and application of a recombinant protein that lacks the collagen-like region of BclA from Bacillus anthracis spores.

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called BclA, which comprises a central collagen-like region (CLR) and a globular C-terminal domain. Here, the entire CLR coding sequence of BclA was removed, and the resulting protein (tBclA) produced in Escherichia coli. The crystallographic structure of tBclA was determined to 1.35 A resolution, and consists of an all-beta structure with a TNF-like jelly fold topology (12 beta-strands which form 2 beta-sheets of five strands each) consistent with previous studies on wild-type BclA. These globular domains are tightly packed into trimeric structures (surface shape complementarity; S (c) = 0.83), demonstrating that formation of the core structure of BclA is independent of the anchoring collagen-like region. A polyclonal antibody raised against tBclA recognized B. anthracis spores directly, and showed little cross-reactivity (<10%) with the spores of the closely related species Bacillus cereus and Bacillus thuringiensis, when compared to two other polyclonal antibodies raised against B. anthracis spore extracts and inactivated spores. The tBclA protein was used to purify a pool of specific antibodies from bovine colostrum whey samples from cows inoculated with the Sterne strain anthrax vaccine, which also showed reactivity with B. anthracis spores. Together, these results demonstrate that tBclA provides a safer and more effective way to the production and purification of antibodies with high binding affinity for B. anthracis spores. Biotechnol. Bioeng. 2008;99: 774-782. (c) 2007 Wiley Periodicals, Inc.

Rapid specific detection and quantification of bacteria and archaea involved in mineral sulfide bioleaching using real-time PCR.

A SybrGreen real-time PCR assay was developed to detect and quantify both total and selected 16S rDNA species of bacteria and archaea involved in the bioleaching of metals from sulfide ores. A set of specific and universal primers based on 16S rDNA sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of Acidianus brierleyi, Sulfolobus sp., Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, Acidithiobacillus caldus, and Leptospirillum ferrooxidans. An artificial sequence based on 16S rDNA was constructed to quantify total 16S rDNA in mixed DNA samples. The real-time PCR assay was further validated using a mixture of 16S rDNA amplicons derived from the six different species, each added at a known amount. Finally, the real-time PCR assay was used to monitor the change of 16S rDNA copies of four bioleaching strains inoculated into chalcopyrite airlift column reactors operated at different temperatures. The growth dynamics of these strains correlated well with the expected effects of temperature in the chalcopyrite-leaching environment. The suitability of this method for monitoring microbial populations in industrial bioleaching environments is discussed.

A potential food-grade cloning vector for Streptococcus thermophilus that uses cadmium resistance as the selectable marker.

A potential food-grade cloning vector, pND919, was constructed and transformed into S. thermophilus ST3-1, a plasmid-free strain. The vector contains DNAs from two different food-approved organisms, Streptococcus thermophilus and Lactococcus lactis. The 5.0-kb pND919 is a derivative of the cloning vector pND918 (9.3 kb) and was constructed by deletion of the 4.3-kb region of pND918 which contained DNA from non-food-approved organisms. pND919 carries a heterologous native cadmium resistance selectable marker from L. lactis M71 and expresses the Cd(r) phenotype in S. thermophilus transformants. With the S. thermophilus replicon derived from the shuttle vector pND913, pND919 is able to replicate in the two S. thermophilus industrial strains tested, ST3-1 and ST4-1. Its relatively high retention rate in S. thermophilus further indicates its usefulness as a potential food-grade cloning vector. To our knowledge, this is the first report of a replicative potential food-grade vector for the industrially important organism S. thermophilus.

Cloning vectors for Streptococcus thermophilus derived from a native plasmid.

A 3.5-kb native plasmid (pND103) was identified in Streptococcus thermophilus ST2-1. Preliminary sequence analysis indicated that pND103 belongs to group I S. thermophilus plasmids. A region of approximately 2 kb appears to contain three components: a plus origin of replication (ori) typical of plasmids that replicate via rolling circle replication; a gene encoding a replication protein (rep); and a gene encoding a small heat shock protein (hsp). pND103 was then used to construct S. thermophilus/Escherichia coli hybrid cloning vectors by ligating different portions of pND103 to an origin-probe vector (pND330) composed of pUC19 and a Gram-positive erythromycin resistance gene. The shuttle vectors (pND913, pND914 and pND915) were successfully introduced back into plasmid-free S. thermophilus ST3-1 as well as to Lactococcus lactis LM0230 and E. coli JM109. Segregational and structural stability study indicated that these vectors can be maintained in these hosts. The results indicated that pND913, pND914 and pND915 are potential shuttle cloning vectors for S. thermophilus.

Genetic and transcriptional analysis of a novel plasmid-encoded copper resistance operon from Lactococcus lactis.

A plasmid-borne copper resistance operon (lco) was identified from Lactococcus lactis subsp. lactis LL58-1. The lco operon consists of three structural genes lcoABC. The predicted products of lcoA and lcoB were homologous to chromosomally encoded prolipoprotein diacylglyceral transferases and two uncharacterized proteins respectively, and the product of lcoC is similar to several multicopper oxidases, which are generally plasmid-encoded. This genetic organization represents a new combination of genes for copper resistance in bacteria. The three genes are co-transcribed from a copper-inducible promoter, which is controlled by lcoRS encoding a response regulator and a kinase sensor. The five genes are flanked by two insertion sequences, almost identical to IS-LL6 from L. lactis. Transposon mutagenesis and subcloning analysis indicated that the three structural genes were all required for copper resistance. Copper assay results showed that the extracellular concentration of copper of L. lactis LM0230 containing the lco operon was significantly higher than that of the host strain when copper was added at concentrations from 2 to 3 mM. The results suggest that the lco operon conferred copper resistance by reducing the intracellular accumulation of copper ions in L. lactis.

Cloning of a gene encoding nisin resistance from Lactococcus lactis subsp. lactis M189 which is transcribed from an extended 210 promoter.

A 56-kb plasmid was identified in Lactococcus lactis subsp. lactis (L. lactis) M189 which encodes resistance to nisin (Nis(R)) following mobilization of the plasmid into L. lactis LM0230. The Nis(R) determinant was localized on a 1.6-kb HindIII fragment by DNA restriction fragment deletion and subcloning. An open reading frame (ORF) of 957 bases was identified by sequence analysis and its transcription start site was mapped by primer extension. The ORF is flanked by two regions which exhibit complete homology to parts of the inverted repeat sequences of IS981 and ISS1T. The promoter for transcription was found to consist of an extended -10 site (TgTGtTATAAT) that lacks a -35 site. Function of the extended -10 promoter was demonstrated by its ability to express the promoterless cat gene from Staphylococcus aureus. Base substitution analysis revealed that the TgTGt extension is essential for promoter efficiency in L. lactis.

Cloning and sequence analysis of a plasmid replicon from Lactococcus lactis subsp. cremoris FG2.

A replication region from one of the Lactococcus lactis subsp. cremoris FG2 plasmids was isolated by cloning of a 4.8-kb XbaI fragment into a replication probe vector and transformation into L. lactis LM0230. A 1.8-kb region within this fragment was sequenced and confirmed by PCR subcloning to encode a functional replicon in LM0230. The replicon consists of an open reading frame encoding a putative replication protein (Rep) of 386 amino acids and a non-coding region (ori) which features several structural motifs typical of other known replication origins, including a 22-bp iteron sequence tandemly repeated three and a half times, a 10-bp direct repeat and two sets of inverted repeats. The ori region could drive replication of its plasmid when supplied with the replication region in-trans. The lack of detectable single-stranded DNA during replication and the existence of extensive homology with other known lactococcal theta replicons strongly suggest that this region encodes a theta-replicating mechanism.