Effect of Antimicrobial Peptide BiF2_5K7K on Contaminated Bacteria Isolated from Boar Semen and Semen Qualities during Preservation and Subsequent Fertility Test on Pig Farm.

The purpose of this study was to determine the impact of an antimicrobial peptide, BiF2_5K7K, on semen quality and bacterial contamination in boar semen doses used for artificial insemination. A key factor affecting semen quality and farm production is bacterial contamination in semen doses. Using antibiotics in a semen extender seems to be the best solution for minimizing bacterial growth during semen preservation. However, concern regarding antibiotic-resistant microorganisms has grown globally. As a result, antimicrobial peptides have emerged as interesting alternative antimicrobial agents to replace the current antibiotics used in semen extenders. BiF2_5K7K is an antimicrobial peptide that can inhibit Gram-negative and Gram-positive bacteria isolated from boar semen and sow vaginal discharge. In this study, ten fresh boar semen samples were collected and diluted with one of two types of semen extender: with (positive control) or without (negative control) an antibiotic (i.e., gentamicin). The semen extender without an antibiotic contained antimicrobial peptide BiF2_5K7K at different concentrations (15.625, 31.25, 62.5, and 125 µg/mL). The samples were stored at 18 °C until use. Semen quality parameters were assessed on days 0, 1, 3, and 5, and the total bacterial count was also evaluated at 0, 24, 36, 48, and 72 h after storage. A fertility test on a pig farm was also performed via sow insemination with a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. No significant difference was found in terms of semen quality on days 0 or 1. On days 3 and 5, the total motility, progressive motility, and viability remained normal in the 15.625 and 31.25 µg/mL groups. However, the sperm parameters decreased starting on day 3 for the 125 µg/mL group and on day 5 for the 62.5 µg/mL group. For total bacterial count at 0, 24, 36, 48, and 72 h, the lowest bacterial count was found in the positive control group, and the highest bacterial count was found in the negative control group compared with the other groups. Comparing antimicrobial peptide groups from 0 to 48 h, the lowest bacterial count was found in the 125 µg/mL group, and the highest bacterial count was found in the 15.625 µg/mL group. For the fertility test, artificial insemination was conducted by using a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. The results showed a superior pregnancy rate, farrowing rate, and total number of piglets born compared with artificial insemination conducted using a commercial extender plus antibiotic. In conclusion, BiF2_5K7K can inhibit bacterial growth in extended boar semen for 24 h, and thereafter, the bacterial count slightly increases. However, the increase in the number of bacterial counts from days 0 to 3 had no negative effect on sperm quality in the positive control, 15.625, or 31.25 µg/mL groups. This indicates that BiF2_5K7K might be an antimicrobial peptide candidate with potential for use as an alternative antimicrobial agent to replace the conventional antibiotic used in boar semen extenders.

Deciphering the Intracellular Action of the Antimicrobial Peptide A11 via an In-Depth Analysis of Its Effect on the Global Proteome of Acinetobacter baumannii.

The potential antimicrobial activity and low propensity to induce the development of bacterial resistance have rendered antimicrobial peptides (AMPs) as novel and ideal candidate therapeutic agents for the treatment of infections caused by drug-resistant pathogenic bacteria. The targeting of bacterial membranes by AMPs has been typically considered their sole mode of action; however, increasing evidence supports the existence of multiple and complementary functions of AMPs that result in bacterial death. An in-depth characterization of their mechanism of action could facilitate further research and development of AMPs with higher potency. The current study employs biophysics and proteomics approaches to unveil the mechanisms underlying the antibacterial activity of A11, a potential candidate AMP, against Acinetobacter baumannii, a leading cause of hospital-acquired infections (HAIs) and consequently, a serious global threat. A11 peptide was found to induce membrane depolarization to a high extent, as revealed by flow cytometry and electron microscopy analyses. The prompt intracellular penetration of A11 peptide, observed using confocal microscopy, was found to occur concomitantly with a very low degree of membrane lysis, suggesting that its mode of action predominantly involves a nonlytic killing mechanism. Quantitative proteomics analysis employed for obtaining insights into the mechanisms underlying the antimicrobial activity of A11 peptide revealed that it disrupted energy metabolism, interfered with protein homeostasis, and inhibited fatty acid synthesis that is essential for cell membrane integrity; all these impacted the cellular functions of A. baumannii. A11 treatment also impacted signal transduction associated with the regulation of biofilm formation, hindered the stress response, and influenced DNA repair processes; these are all crucial survival mechanisms of A. baumannii. Additionally, robust antibacterial activity was exhibited by A11 peptide against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of A. baumannii; moreover, A11 peptide exhibited synergy with levofloxacin and minocycline as well as low propensity for inducing resistance. Taken together, the findings emphasize the therapeutic potential of A11 peptide as an antibacterial agent against drug-resistant A. baumannii and underscore the need for further investigation.

The Effects of Different Antimicrobial Peptides (A-11 and AP19) on Isolated Bacteria from Fresh Boar Semen and Semen Quality during Storage at 18 °C.

Antibiotic resistance (AMR) is a major public health concern. Antimicrobial peptides (AMPs) could be an alternative to conventional antibiotics. The purpose of this research was to investigate the antimicrobial ability of the synthetic AMPs (i.e., A-11 and AP19) on the most frequently isolated bacteria in boar semen and their effect on extended boar semen quality during storage. We tested the antimicrobial effect of A-11 and AP19 at different concentrations and compared them with gentamicin for inhibiting the growth of E. coli, Pseudomonas aeruginosa and Proteus mirabilis that were isolated from fresh boar semen. In order to evaluate the effect of AMP on semen qualities on days 0, 1, 3, and 5 after storage at 18 °C, seven fresh boar semen samples were collected, diluted with semen extender with antibiotic (i.e., gentamicin at 200 µg/mL, positive control) or without (negative control), and semen extender contained only A-11 or AP19 at different concentrations (i.e., 62.50, 31.25, and 15.625 µg/mL). The total bacterial count was also measured at 0, 24, 36, 48, and 72 h after storage. Comparable to gentamicin, both A-11 and AP19 inhibited the growth of E. coli, Pseudomonas aeruginosa, and Proteus mirabilis at 62.50, 31.25, and 15.625 µg/mL, respectively. Comparing the total bacterial count at 0, 24, 36, 48 and 72 h after storage, the lowest total bacterial concentration was found in the positive control group (p < 0.05), and an inferior total bacterial concentration was found in the treatment groups than in the negative control. On day 1, there is a lower percentage of all sperm parameters in the AP19 group at a concentration of 62.50 µg/mL compared with the other groups. On day 3, the highest percentage of all sperm parameters was found in the positive control and A-11 at a concentration of 31.25 µg/mL compared with the other groups. The AP19 group at 62.5 µg/mL constantly yielded inferior sperm parameters. On day 5, only A-11 at a concentration of 15.625 µg/mL showed a total motility higher than 70%, which is comparable to the positive control. A-11 and AP19 showed antimicrobial activity against E. coli, Pseudomonas aeruginosa and Proteus mirabilis isolated from boar semen. Considering their effect on semen quality during storage, these antimicrobial peptides are an alternative to conventional antibiotics used in boar semen extenders. Nevertheless, the utilization of these particular antimicrobial peptides relied on the concentration and duration of storage.

Dual-function antimicrobial-antibiofilm peptide hybrid to tackle biofilm-forming Staphylococcus epidermidis.

BACKGROUND: Due to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication. METHODS: A novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area. RESULTS: BiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area. CONCLUSIONS: Due to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis.

The Effect of Antimicrobial Peptide (PA-13) on Escherichia coli Carrying Antibiotic-Resistant Genes Isolated from Boar Semen.

A major global public health concern is antimicrobial resistance (AMR). Antimicrobial peptides (AMPs) are a potentially appropriate replacement for conventional antibiotics. The purpose of this research was to investigate the potential of the antimicrobial peptide PA-13, a synthetic AMP with 13 amino acids, to inhibit E. coli isolated from boar semen expressing antibiotic-resistant genes, as well as to determine the mechanism of action of this antimicrobial peptide on the bacterial membrane. The effectiveness of the bacterial inhibitory activity of PA-13 was tested at different concentrations by two fold serial dilutions in the range 0.488-500 µg/mL using the MIC and MBC methods. The impact of PA-13 on the bacterial membrane was examined at different concentrations of 0×, 0.5×, 1×, 2× and 4× of MIC using DNA leakage assay and electron microscopy. The PA-13 antibacterial activity result exhibited the same MIC and MBC values at a concentration of 15.625 µg/mL. When comparing DNA leakage at different MIC values, the results revealed that the maximum amount of DNA concentration was found two and three hours after incubation. For the results of SEM and TEM, the bacterial membrane disruption of this E. coli was found in the PA-13-treated group when compared with the negative control. In conclusion, synthetic PA-13 with its antibacterial properties is an alternative antimicrobial peptide to antibiotics in the pig industry.

The bactericidal and antibiofilm effects of a lysine-substituted hybrid peptide, CM-10K14K, on biofilm-forming Staphylococcus epidermidis.

Staphylococci, notably biofilm-forming Staphylococcus epidermidis, have been recognized as global nosocomial pathogens in medical device-related infections. Their potential to attach to and form biofilm on indwelling catheters are significant factors impeding conventional treatment. Due to their extensive antimicrobial and antibiofilm actions, antimicrobial peptides (AMPs) have attracted interest as promising alternative compounds for curing difficult-to-treat, biofilm-forming bacterial infections. Cecropin A-melittin or CM, a well-known hybrid peptide, exhibits broad-spectrum antimicrobial activity, however it also possesses high toxicity. In the current study, a series of hybrid CM derivatives was designed using an amino acid substitution strategy to explore potential antibacterial and antibiofilm peptides with low toxicity. Among the derivatives, CM-10K14K showed the least hemolysis along with potent antibacterial activity against biofilm-forming S. epidermidis (MICs = 3.91 µg/mL) and rapid killing after 15 min exposure (MBCs = 7.81 µg/mL). It can prevent the formation of S. epidermidis biofilm and also exhibited a dose-dependent eradication activity on mature or established S. epidermidis biofilm. In addition, it decreased the development of biofilm by surviving bacteria, and formation of biofilm on the surface of CM-10K14K-impregnated catheters. Released CM-10K14K decreased planktonic bacterial growth and inhibited biofilm formation by S. epidermidis in a dose-dependent manner for 6 and 24 h post-exposure. Impregnation of CM-10K14K prevented bacterial attachment on catheters and thus decreased formation of extensive biofilms. SEM images supported the antibiofilm activity of CM-10K14K. Flow cytometry analysis and TEM images demonstrated a membrane-active mechanism of CM-10K14K, inducing depolarization and permeabilization, and subsequent membrane rupture leading to cell death. The presence of an interaction with bacterial DNA was verified by gel retardation assay. These antibacterial and antibiofilm activities of CM-10K14K suggest its potential application to urinary catheters for prevention of biofilm-forming colonization or for treatment of medical devices infected with S. epidermidis.

Palm Kernel Meal Protein Hydrolysates Enhance Post-Thawed Boar Sperm Quality.

Boar sperm is sensitive to particular conditions during cryopreservation, resulting in an extreme reduction in fertilizing ability due to damage to the sperm membranes. PKMPH contains bioactive peptides that have antioxidant and antimicrobial activities. There is no information on the use of palm-kernel-meal-derived bioactive peptides for boar semen cryopreservation. This study aimed to examine the effects of bioactive peptides from PKMPH on post-thawed boar sperm quality. Boar semen ejaculates (n = 17) were collected and divided into six equal aliquots based on PKMPH concentrations (0, 1.25, 2.5, 5, 10, and 15 µg/mL) in a freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, the frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm motility using a computer-assisted sperm analyzer and for sperm viability, acrosome integrity, mitochondrial function, and lipid peroxidation by measuring the level of malondialdehyde (MDA). The results demonstrate that the supplementation of PKMPH with 2.5 µg/mL afforded superior post-thawed sperm qualities, such as increased total motility, viability, acrosome integrity, and mitochondrial function by 10.7%, 12.3%, 18.3%, and 12.7%, respectively, when compared to the control group. PKMPH at a concentration of 2.5 µg/mL showed the lowest level of MDA (40.6 ± 2.0 µMol/L) compared to the other groups. In conclusion, adding PKMPH peptides at 2.5 µg/mL to the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in higher post-thawed sperm quality.

Development and Validation of an Efficient Multiplex PCR Assay for Simultaneous Detection of Six Common Foodborne Pathogens and Hygiene Indicators.

Microbial contamination in foods could lead to illnesses and substantial losses in both food industry and public health sectors. Rapid detection of microbial hazards (i.e., pathogens, hygiene indicator microorganisms) can accelerate surveillance and diagnostic processes reducing transmission and minimizing undesirable consequences. This study developed a multiplex PCR (m-PCR) for the detection of six common foodborne pathogens and hygiene indicators using specific primers for uidA of Escherichia coli, stx2 of Escherichia coli O157:H7, invA of Salmonella spp., int of Shigella spp., ntrA of Klebsiella pneumoniae, and ail of Yersinia enterocolitica and Yersinia pseudotuberculosis. Sensitivity of the m-PCR was 100 fg or ∼20 bacterial cells. Each primer set amplified only the targeted strain, and specificity was demonstrated by lack of nonspecific bands with DNA from 12 other bacterial strains. Following ISO 16140-2:2016, the relative limit of detection of the m-PCR was comparable to that of the gold-standard method; however, the processing time was five times faster. The m-PCR was applied to detect the six pathogens in 100 natural samples (50 pork meat and 50 local fermented food samples) and compared to results of the gold-standard method. Positive cultures for Klebsiella, Salmonella, and E. coli were 66%, 82%, and 88%, respectively, of meat samples and 78%, 26%, and 56%, respectively, of fermented food samples. Escherichia coli O157:H7, Shigella, and Yersinia were not detected in any of the samples by both standard and m-PCR methods. The developed m-PCR assay showed comparable results with the traditional culture technique proving its rapid and reliable detection of six foodborne pathogens and hygiene indicators in food.

A novel designed membrane-active peptide for the control of foodborne Salmonella enterica serovar Typhimurium.

The main cause of non-typhoidal Salmonella (NTS) infection in humans is ingestion of contaminated animal-derived foods such as eggs, poultry and dairy products. These infections highlight the need to develop new preservatives to increase food safety. Antimicrobial peptides (AMPs) have the potential to be further developed as food preservative agents and join nisin, the only AMP currently approved, for use as a preservative in food. Acidocin J1132ß, a bacteriocin produced by probiotic Lactobacillus acidophilus, displays no toxicity to humans, however it exhibits only low and narrow-spectrum antimicrobial activity. Accordingly, four peptide derivatives (A5, A6, A9, and A11) were modified from acidocin J1132ß by truncation and amino acid substitution. Among them, A11 showed the most antimicrobial activity, especially against S. Typhimurium, as well as a favorable safety profile. It tended to form an α-helix structure upon encountering negatively charged-mimicking environments. A11 caused transient membrane permeabilization and killed bacterial cells through membrane depolarization and/or intracellular interactions with bacterial DNA. A11 maintained most of its inhibitory effects when heated, even when exposed to temperatures up to 100 °C. Notably, it inhibited drug-resistant S. Typhimurium and its monophasic variant strains. Furthermore, the combination of A11 and nisin was synergistic against drug-resistant strains in vitro. Taken together, this study indicated that a novel antimicrobial peptide derivative (A11), modified from acidocin J1132ß, has the potential to be a bio-preservative to control S. Typhimurium contamination in the food industry.

Novel D-form of hybrid peptide (D-AP19) rapidly kills Acinetobacter baumannii while tolerating proteolytic enzymes.

Antimicrobial peptides (AMPs) are being developed as potent alternative treatments to conventional antibiotics which are unlikely to induce bacterial resistance. They can be designed and modified to possess several druggable properties. We report herein a novel hybrid peptide of modified aurein (A3) and cathelicidin (P7), or A3P7, by a flipping technique. It exhibited potent antibacterial activity against both Gram-negative and -positive pathogenic bacteria but had moderate hemolytic activity. To reduce the sequence length and toxicity, C-terminal truncation was serially performed and eight truncated derivatives (AP12-AP19) were obtained. They had significantly less hemolytic activity while preserving antibacterial activity. Secondary structures of the candidate peptides in environments simulating bacterial membranes (30 mM SDS and 50% TFE), determined by CD spectroscopy, showed α-helical structures consistent with predicted in silico 3D structural models. Among the peptides, AP19 demonstrated the best combination of broad-spectrum antibacterial activity (including toward Acinetobacter baumannii) and minimal hemolytic and cytotoxic activities. A D-form peptide (D-AP19), in which all L-enantiomers were substituted with the D-enantiomers, maintained antibacterial activity in the presence of pepsin, trypsin, proteinase K and human plasma. Both isomers exhibited potent antibacterial activity against multi-drug (MDR) and extensively-drug resistant (XDR) clinical isolates of A. baumannii comparable to the traditional antibiotic, meropenem. D-AP19 displayed rapid killing via membrane disruption and leakage of intracellular contents. Additionally, it showed a low tendency to induce bacterial resistance. Our work suggested that D-AP19 could be further optimized and developed as a novel compound potentially for fighting against MDR or XDR A. baumannii.

Biopolymer Nano-Network for Antimicrobial Peptide Protection and Local Delivery.

Antimicrobial resistance (AMR) develops when bacteria no longer respond to conventional antimicrobial treatment. The limited treatment options for resistant infections result in a significantly increased medical burden. Antimicrobial peptides offer advantages for treatment of resistant infections, including broad-spectrum activity and lower risk of resistance development. However, sensitivity to proteolytic cleavage often limits their clinical application. Here, a moldable and biodegradable colloidal nano-network is presented that protects bioactive peptides from enzymatic degradation and delivers them locally. An antimicrobial peptide, PA-13, is encapsulated electrostatically into positively and negatively charged nanoparticles made of chitosan and dextran sulfate without requiring chemical modification. Mixing and concentration of oppositely charged particles form a nano-network with the rheological properties of a cream or injectable hydrogel. After exposure to proteolytic enzymes, the formed nano-network loaded with PA-13 eliminates Pseudomonas aeruginosa during in vitro culture and in an ex vivo porcine skin model while the unencapsulated PA-13 shows no antibacterial effect. This demonstrates the ability of the nano-network to protect the antimicrobial peptide in an enzyme-challenged environment, such as a wound bed. Overall, the nano-network presents a useful platform for antimicrobial peptide protection and delivery without impacting peptide bioactivity.

A Mitochondria-Penetrating Peptide Exerts Potent Anti-Plasmodium Activity and Localizes at Parasites' Mitochondria.

Mitochondria are considered a novel drug target as they play a key role in energy production and programmed cell death of eukaryotic cells. The mitochondria of malaria parasites differ from those of their vertebrate hosts, contributing to the drug selectivity and the development of antimalarial drugs. (Fxr)3, a mitochondria-penetrating peptide or MPP, entered malaria-infected red cells without disrupting the membrane and subsequently killed the blood stage of P. falciparum parasites. The effects were more potent on the late stages than on the younger stages. Confocal microscopy showed that the (Fxr)3 intensely localized at the parasite mitochondria. (Fxr)3 highly affected both the lab-strain, chloroquine-resistant K1, and freshly isolated malaria parasites. (Fxr)3 (1 ng/mL to 10 µg/mL) was rarely toxic towards various mammalian cells, i.e., mouse fibroblasts (L929), human leukocytes and erythrocytes. At a thousand times higher concentration (100 µg/mL) than that of the antimalarial activity, cytotoxicity and hemolytic activity of (Fxr)3 were observed. Compared with the known antimalarial drug, atovaquone, (Fxr)3 exhibited more rapid killing activity. This is the first report on antimalarial activity of (Fxr)3, showing localization at parasites' mitochondria.

Effective inhibition of Clostridioides difficile by the novel peptide CM-A.

Clostridioides difficile infection is the most common cause of nosocomial and antibiotic-associated diarrhea. C. difficile treatment is increasingly likely to fail, and the recurrence rate is high. Antimicrobial peptides are considered an alternative treatment for many infectious diseases, including those caused by antibiotic resistant bacteria. In the present study, we identified a CM peptide, a hybrid of cecropin A and melittin, and its derivative which possesses potent antimicrobial activity against C. difficile strain 630. CM peptide exhibited antibacterial activity with minimum inhibitory concentration of 3.906 µg/ml (2.21 µM). A modified derivative of CM, CM-A, exhibited even greater activity with a minimum inhibitory concentration of 1.953 µg/ml (1.06 µM) and a minimum bactericidal concentration of 7.8125 µg/ml (4.24 µM), which indicates that CM-A peptide is more efficient than its parent peptide. A fluorescence-activated cell sorter analysis revealed that the membrane of C. difficile 630 could be an important target for CM-A. This peptide induced high levels of cell depolarization and cell permeability on C. difficile cell membrane. Moreover, electron microscopy imaging showed that CM-A interferes with the C. difficile cell membrane. Hence, the antimicrobial peptide CM-A may represent a promising novel approach for the treatment of C. difficile infections.

Enzymatic characterization and structure-function relationship of two chitinases, LmChiA and LmChiB, from Listeria monocytogenes.

Listeria monocytogenes possesses two chitinases (LmChiA and LmChiB) belonging to glycoside hydrolase family 18 (GH18). In this study, two chitinase genes (lmchiA and lmchiB) from L. monocytogenes 10403S were cloned and their biochemical characteristics were studied. Using colloidal chitin as substrate, both chitinases exhibited maximum catalytic activity at pH 6-7 with optimum temperature at 50 °C. Their activities were stable over broad pH (3-10) and temperature (10-50 °C) ranges. Kinetic analysis using [4NP-(GlcNAc)2] as substrate indicated that LmChiB had an approximately 4-fold lower K m and 2-fold higher k cat than LmChiA, suggesting that the catalytic specificity and efficiency of LmChiB were greater than those of LmChiA. LmChiA and LmChiB showed the same reactivity toward oligomeric substrates and exhibited both non-processive endo-acting and processive exo-acting (chitobiosidase) activity on colloidal chitin, chitin oligosaccharides and 4-nitrophenyl substrates. Structure-based sequence alignments and homology modeling of the catalytic domains revealed that both chitinases consisted of an (α/ß)8 TIM barrel fold with a conserved DXDXE motif. The key residues involved in the substrate hydrolysis were conserved with other bacterial chitinases. The site-directed mutagenesis of conserved Asp and Glu residues in DXDXE motif of both chitinases significantly reduced the chitinolytic activity toward colloidal chitin substrate and revealed their critical role in the catalytic mechanism. LmChiA and LmChiB might have potential in chitin waste utilization and biotechnological applications.

A novel, rationally designed, hybrid antimicrobial peptide, inspired by cathelicidin and aurein, exhibits membrane-active mechanisms against Pseudomonas aeruginosa.

Antimicrobial peptides (AMPs) are promising alternatives to classical antibiotics for the treatment of drug-resistant infections. Due to their versatility and unlimited sequence space, AMPs can be rationally designed by modulating physicochemical determinants to favor desired biological parameters and turned into novel therapeutics. In this study, we utilized key structural and physicochemical parameters, in combination with rational engineering, to design novel short α-helical hybrid peptides inspired by the well-known natural peptides, cathelicidin and aurein. By comparing homologous sequences and abstracting the conserved residue type, sequence templates of cathelicidin (P0) and aurein (A0) were obtained. Two peptide derivatives, P7 and A3, were generated by amino acid substitution based on their residue composition and distribution. In order to enhance antimicrobial activity, a hybrid analog of P7A3 was designed. The results demonstrated that P7A3 had higher antibacterial activity than the parental peptides with unexpectedly high hemolytic activity. Strikingly, C-terminal truncation of hybrid peptides containing only the α-helical segment (PA-18) and shorter derivatives confer potent antimicrobial activity with reduced hemolytic activity in a length-dependent manner. Among all, PA-13, showed remarkable broad-spectrum antibacterial activity, especially against Pseudomonas aeruginosa with no toxicity. PA-13 maintained antimicrobial activity in the presence of physiological salts and displayed rapid binding and penetration activity which resulted in membrane depolarization and permeabilization. Moreover, PA-13 showed an anti-inflammatory response via lipopolysaccharide (LPS) neutralization with dose-dependent, inhibiting, LPS-mediated Toll-like receptor activation. This study revealed the therapeutic potency of a novel hybrid peptide, and supports the use of rational design in development of new antibacterial agents.

A Thermostable, Modified Cathelicidin-Derived Peptide With Enhanced Membrane-Active Activity Against Salmonella enterica serovar Typhimurium.

Foodborne illness caused by consumption of food contaminated with Salmonella is one of the most common causes of diarrheal disease and affects millions of people worldwide. The rising emergence and spread of antimicrobial resistance, especially in some serotypes of Salmonella, has raised a great awareness of public health issues worldwide. To ensure safety of the food processing chain, the development of new food preservatives must be expedited. Recently, thermal- and pH-stable antimicrobial peptides have received much attention for use in food production, and represent safe alternatives to chemical preservatives. A 12-mer cathelicidin-derived, α-helical cationic peptide, P7, displayed rapid killing activity, against strains of drug-resistant foodborne Salmonella enterica serovar Typhimurium and its monophasic variant (S. enterica serovar 4,5,12:i:-) and had minimal toxicity against mouse fibroblast cells. P7 tended to form helical structure in the membrane-mimic environments as evaluated by circular dichroism (CD) spectroscopy. The action mode of P7 at the membrane-level was affirmed by the results of flow cytometry, and confocal, scanning and transmission electron microscopy. P7 killed bacteria through binding to bacterial membranes, penetration and the subsequent accumulation in S. enterica serovar Typhimurium cytoplasm. This induced membrane depolarization, permeabilization, and sequential leakage of intracellular substances and cell death. Except for sensitivity to proteolytic digestive enzymes, P7 maintained its inhibitory activity against S. enterica serovar Typhimurium in the presence of different conditions [various salts, extreme pHs and heat (even at 100°C)]. Moreover, the peptide is unlikely to induce bacterial resistance in vitro. Taken together, this study demonstrated that the membrane-permeabilizing P7 peptide has much potential as a new antimicrobial agent for use in food processing and preservation.

Structural and biological features of a novel plant defensin from Brugmansia x candida.

Data from both the laboratory and clinic in the last decade indicate that antimicrobial peptides (AMPs) are widely regarded as potential sources of future antibiotics owing to their broad-spectrum activities, rapid killing, potentially low-resistance rate and multidirectional mechanisms of action compared to conventional antibiotics. Defensins, a prominent family of AMPs, have been found in a wide range of organisms including plants. Thailand is a rich source of plants including medicinal plants used therapeutically, however there is no report of defensin from among these plants. In this study, a novel plant defensin gene, BcDef, was successfully cloned from Brugmansia x candida (Bc). BcDef cDNA was 237 bp in length, encoding 78 amino acids with a putative 31-amino acid residue signal peptide at the N-terminal followed by the mature sequence. BcDef shared high sequence identity (78-85%) with Solanaceae defensins and belonged to the class I plant defensins. From homology modeling, BcDef shared a conserved triple stranded ß-sheet (ß1-ß3) and one α-helix (α1) connected by a loop (L1-L3). BcDef1 peptide, designed from the γ-core motifs of BcDef located in loop 3, showed antibacterial activity against both Gram-positive and Gram-negative pathogens with the lowest MIC (15.70 µM) against Staphylococcus epidermidis. This peptide affected cell membrane potential and permeability, and caused cell membrane disruption. Moreover, BcDef1 also exhibited antioxidant activity and showed low cytotoxicity against mouse fibroblast L929 cells. These findings may provide an opportunity for developing a promising antibacterial agent for medical application in the future.

Prevalence and Risk Factors for Blastocystis Infection Among Children and Caregivers in a Child Care Center, Bangkok, Thailand.

In September 2009, a cross-sectional study was conducted to evaluate parasitic infections in a child care center in Khlong Toei, Bangkok, Thailand. Of 503 children and staff members, 258 (51.3%) stool samples and questionnaires were obtained. The most common parasitic infection was Blastocystis sp. (13.6%). Blastocystis sp. subtype 3 was predominantly found (80.0%), followed by subtypes 2 (12.0%) and 1 (8.0%). The prevalence of Blastocystis infection varied among different age groups. The prevalence of Blastocystis infection in non-HIV-infected children aged < 10 and 10-19 years were 14.5% and 10.3%, respectively, which were not significantly different. All 31 HIV-infected children were not infected with Blastocystis sp. The most likely reason could be the result of properly using prevention measures for this specific group.

Colicin type 7 produced by majority of Shigella sonnei isolated from Thai patients with diarrhoea

Thirty one out of 153 strains of Shigella sonnei isolated from Thai patients with diarrhoea showed antibacterial activity against S. sonnei by agar well diffusion method. All of them harbor plasmids with the genetic determination of colicin type 7 (Js) gene but without colicin E and colicin U gene. The PCR product obtained from strain 35/44 was shown to be the gene for colicin type 7 lytic protein (cja). The partially purified bacteriocin (PPB) containing colicin type 7 of strain 35/44 was prepared and used for characterization. The antibacterial activity of PPB against a total of 17 selected Gram-positive and Gram-negative bacteria was tested. It was found that PPB of strain 35/44 was active against E. coli O157, S. sonnei and S. boydii. The sensitivity of PPB from this strain to proteinase K, trypsin and α-chymotrypsin suggests the proteinaceous nature of these antimicrobial substances. Therefore, this isolated bacterium can be regarded as bacteriocin producing bacteria. The bacteriocin produced by this isolated S. sonnei was heat stable as evidenced by its ability to maintain the activity at 80 °C for 60 min. In addition, it was stable within a wide range of pH (3-9). The molecular weight of colicin type 7 from isolated S. sonnei strain 35/44 analyzed by SDS-PAGE was 54.4 kDa composing of at least five subunits. It is to our knowledge; the first report of Thai patients with diarrhoea that S. sonnei isolated from them contained colicin type 7.