Distribution of antimicrobial‐resistant lactic acid bacteria in natural cheese in Japan

To determine and compare the extent of contamination caused by antimicrobial‐resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P = 0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm‐made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm‐made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to > 512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial‐resistant LAB in imported and Japanese farm‐made cheeses on the Japanese market, but not in Japanese commercial cheeses.     

Conformation control of the histidine kinase BceS of Bacillus subtilis by its cognate ABC‐transporter facilitates need‐based activation of antibiotic resistance

Bacteria closely control gene expression to ensure optimal physiological responses to their environment. Such careful gene expression can minimize the fitness cost associated with antibiotic resistance. We previously described a novel regulatory logic in Bacillus subtilis enabling the cell to directly monitor its need for detoxification. This cost‐effective strategy is achieved via a two‐component regulatory system (BceRS) working in a sensory complex with an ABC‐transporter (BceAB), together acting as a flux‐sensor where signaling is proportional to transport activity. How this is realized at the molecular level has remained unknown. Using experimentation and computation we here show that the histidine kinase is activated by piston‐like displacements in the membrane, which are converted to helical rotations in the catalytic core via an intervening HAMP‐like domain. Intriguingly, the transporter was not only required for kinase activation, but also to actively maintain the kinase in its inactive state in the absence of antibiotics. Such coupling of kinase activity to that of the transporter ensures the complete control required for transport flux‐dependent signaling. Moreover, we show that the transporter likely conserves energy by signaling with sub‐maximal sensitivity. These results provide the first mechanistic insights into transport flux‐dependent signaling, a unique strategy for energy‐efficient decision making.     

Screening and characterization of a novel high‐efficiency tumor‐homing cell‐penetrating peptide from the buffalo cathelicidin family

Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell‐penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo‐derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell‐penetrating antimicrobial peptides, including the well‐known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC‐7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC‐5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia‐derived type‐I ribosome‐inactivating protein MAP 30, and the cytotoxicity of the MAP 30‐CAT fusion protein in the tumor cell line SMMC‐7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30‐CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC‐5 was approximately twofold higher than the value for SMMC‐7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38‐fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor‐homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.     

Diversity of the Lactobacillus group in breast milk and vagina of healthy women and potential role in the colonization of the infant gut

AIMS: To evaluate the diversity of the Lactobacillus group in breast milk and the vagina of healthy women and understand their potential role in the infant gut colonization using the 16S rRNA gene approaches. METHODS AND RESULTS: Samples of breast milk, vaginal swabs and infant faeces were aseptically collected from five mothers whose neonates were born by vaginal delivery and another five that had their babies by caesarean section. After polymerase chain reaction (PCR) amplification using Lactobacillus group-specific primers, amplicons were analysed by denaturing gradient gel electrophoresis (DGGE). Clone libraries were constructed to describe the Lactobacillus group diversity. DGGE fingerprints were not related to the delivery method. None of the species detected in vaginal samples were found in breast milk-derived libraries and only few were detected in infant faeces. CONCLUSIONS: The bacterial composition of breast milk and infant faeces is not related to the delivery method. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been suggested that neonates acquire lactobacilli by oral contamination with vaginal strains during delivery; subsequently, newborns would transmit such bacteria to the breast during breastfeeding. However, our findings confirm, at the molecular level that in contrast to the maternal vagina, breast milk seems to constitute a good source of lactobacilli to the infant gut.     

Microbial production of conjugated γ‐linolenic acid from γ‐linolenic acid by Lactobacillus plantarum AKU 1009a

Aims: Optimal production conditions of conjugated γ‐linolenic acid (CGLA) from γ‐linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. Methods and Results: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0·03% (w/v) α‐linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml^?1γ‐linolenic acid as a substrate in 5 ‐ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml^?1 dry cells] as the catalysts produced 8·8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis‐6,cis‐9,trans‐11‐octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis‐6,trans‐9,trans‐11‐octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. Conclusion: The practical process of CGLA production from γ‐linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. Significance and Impact of the Study: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.     

Effect of milk fermented with a Lactobacillus helveticus R389(+) proteolytic strain on the immune system and on the growth of 4T1 breast cancer cells in mice

Previous studies on a murine model have demonstrated that the administration of Lactobacillus helveticus and Lactobacillus casei inhibits the development of fibrosarcoma and colon carcinoma, respectively. The aim of this work was to study the beneficial effects of the consumption of milk fermented by L. helveticus on a murine model for mammary carcinoma. Female BALB/c mice were challenged by a single subcutaneous injection of tumoral cells (American Type Culture Collection 4T1) in the left mammary gland. Prior to tumour injection, mice were fed for two, five or seven consecutive days with fermented milk. The following factors were monitored for 2 months: rate of tumour development, histological studies, apoptosis, phagocytic index, peritoneal macrophages, determination of beta-glucuronidase enzyme in peritoneal macrophages, determination of gamma-interferon (INFgamma) and tumour necrosis factor-alpha (TNF-alpha) in blood serum, determination of CD4+, CD8+, interleukin-6 (IL-6), IL-10, TNF-alpha and INFgamma by immunoperoxidase, and measurement of beta-glucuronidase activity in intestinal fluid. The administration of L. helveticus delayed the development of the tumour in all cases, a 2- or 7-day feeding period being most effective. This work demonstrates that milk fermented with L. helveticus decreases the growth rate of mammary tumours. The effect was mediated by increased apoptosis and decreased production of pro-inflammatory cytokines, in particular IL-6, implicated in oestrogen synthesis.     

Isolation and basic characterization of a β‐glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture

Aims: To study glycosidase activities of a Lactobacillus brevis strain and to isolate an intracellular β‐glucosidase from this strain. Methods and Results: Lactic acid bacteria (LAB) isolated from a commercially available starter culture preparation for malolactic fermentation were tested for β‐glycosidase activities. A strain of Lact. brevis showing high intracellular β‐d ‐glucosidase, β‐d ‐xylosidase and α‐l ‐arabinosidase activities was selected for purification and characterization of its β‐glucosidase. The pure glucosidase from Lact. brevis has also side activities of xylosidase, arabinosidase and cellobiosidase. It is a homotetramer of 330 kDa and has an isoelectric point at pH 3·5. The K_m for p‐nitrophenyl‐β‐d ‐glucopyranoside and p‐nitrophenyl‐β‐d ‐xylopyranoside is 0·22 and 1·14 mmol l^?1, respectively. The β‐glucosidase activity was strongly inhibited by gluconic acid δ‐lactone, partially by glucose and gluconate, but not by fructose. Ethanol and methanol were found to increase the activity up to twofold. The free enzyme was stable at pH 7·0 (t_1/2 = 50 day) but not at pH 4·0 (t_1/2 = 4 days). Conclusions: The β‐glucosidase from Lact. brevis is widely different to that characterized from Lactobacillus casei ( Coulon et al. 1998 ) and Lactobacillus plantarum ( Sestelo et al. 2004 ). The high tolerance to fructose and ethanol, the low inhibitory effect of glucose on the enzyme activity and the good long‐term stability could be of great interest for the release of aroma compounds during winemaking. Significance and Impact of the study: Although the release of aroma compounds by LAB has been demonstrated by several authors, little information exists on the responsible enzymes. This study contains the first characterization of an intracellular β‐glucosidase isolated from a wine‐related strain of Lact. brevis.     

Analogues of the frog skin peptide alyteserin‐2a with enhanced antimicrobial activities against Gram‐negative bacteria

The emergence of strains of multidrug‐resistant Gram‐negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin‐2a (ILGKLLSTAAGLLSNL.NH₂) is a cationic, α‐helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin‐2a while maintaining amphipathicity by the substitution Gly¹¹→ Lys enhanced the potency against both Gram‐negative and Gram‐positive bacteria by between fourfold and 16‐fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC₅₀ = 24 µm ). Antimicrobial potency was increased further by the additional substitution Ser⁷→Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC₅₀ = 38 µm ). However, the peptide containing d ‐lysine at positions 7 and 11 showed high potency against a range of Gram‐negative bacteria, including multidrug‐resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µm ) but appreciably lower haemolytic activity (LC₅₀ = 185 µm ) and cytotoxicity against A549 human alveolar basal epithelial cells (LC₅₀ = 65 µm ). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Localization of the anti‐cancer peptide EGFR‐lytic hybrid peptide in human pancreatic cancer BxPC‐3 cells by immunocytochemistry

Cationic lytic‐type peptides have been studied for clinical application in various infections and cancers, but their functional cellular mechanisms remain unclear. We generated anti‐cancer epithelial growth factor receptor (EGFR)‐lytic hybrid peptide, a 32‐amino‐acid peptide composed of an EGFR‐binding sequence and lytic sequence. In this study, we investigated the distribution of EGFR‐lytic hybrid peptide in BxPC‐3 human pancreatic cancer cells by an immunocytochemical (ICC) method. Distribution of EGFR protein expression was unchanged after treatment with EGFR‐lytic peptide compared with non‐treated cells. In confocal laser scanning microscopy, immunostaining of EGFR‐lytic peptide was observed in the cytoplasm, mostly in the form of granules. Some staining was also localized on the mitochondrial membrane. At the ultrastructure level, cells treated with EGFR‐lytic peptide had a low electron density, disappearance of microvilli, and swollen mitochondria. Fragments of cell membrane were also observed in the proximity of the membrane. In immunoelectron microscopy, EGFR‐lytic peptide was observed in the cell membrane and cytoplasm. A number of granules were considered swollen mitochondria. Activation of the caspase pathway as a result of mitochondrial dysfunction was also examined to determine the cytotoxic activity of EGFR‐lytic peptide; however, no effect on cell death after EGFR‐lytic treatment was observed, and moreover, apoptosis was not found to play a critical role in the cell death mechanism. These results suggest that EGFR‐lytic peptide is localized on cell and mitochondrial membranes, with disintegration of the cell membrane contributing mainly to cell death. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Enhanced expression of cysteine‐rich antimicrobial peptide snakin‐1 in Escherichia coli using an aggregation‐prone protein coexpression system

Snakin‐1 (SN‐1) is a cysteine‐rich plant antimicrobial peptide and the first purified member of the snakin family. SN‐1 shows potent activity against a wide range of microorganisms, and thus has great biotechnological potential as an antimicrobial agent. Here, we produced recombinant SN‐1 in Escherichia coli by a previously developed coexpression method using an aggregation‐prone partner protein. Our goal was to increase the productivity of SN‐1 via the enhanced formation of insoluble inclusion bodies in E. coli cells. The yield of SN‐1 by the coexpression method was better than that by direct expression in E. coli cells. After refolding and purification, we obtained several milligrams of functionally active SN‐1, the identity of which was verified by MALDI‐TOF MS and NMR studies. The purified recombinant SN‐1 showed effective antimicrobial activity against test organisms. Our studies indicate that the coexpression method using an aggregation‐prone partner protein can serve as a suitable expression system for the efficient production of functionally active SN‐1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1520–1528, 2017     

Effects of dimerization of the cell‐penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action

The cell‐penetrating peptide Tat (48–60) (GRKKRRQRRRPPQ) derived from HIV‐1 Tat protein showed potent antibacterial activity (MIC: 2–8 µM ). To investigate the effect of dimerization of Tat (48–60) analog, [Tat(W): GRKKRRQRRRPWQ‐NH₂], on antimicrobial activity and mechanism of bactericidal action, its dimeric peptides, di‐Tat(W)‐C and di‐Tat(W)‐K, were synthesized by a disulfide bond linkage and lysine linkage of monomeric Tat(W), respectively. From the viewpoint of a weight basis and the monomer concentration, these dimeric peptides displayed almost similar antimicrobial activity against six bacterial strains tested but acted more rapidly against Staphylococcus aureus on kinetics of bactericidal activity, compared with monomeric Tat(W). Unlike monomeric Tat(W), these dimeric peptides significantly depolarized the cytoplasmic membrane of intact S. aureus cells at MIC and induced dye leakage from bacterial‐membrane‐mimicking egg yolk L ‐α‐phosphatidylethanolamine/egg yolk L ‐α‐phosphatidyl‐DL ‐glycerol (7:3, w/w) vesicles. Furthermore, these dimeric peptides were less effective to translocate across lipid bilayers than monomeric Tat(W). These results indicated that the dimerization of Tat analog induces a partial change in the mode of its bactericidal action from intracellular target mechanism to membrane‐targeting mechanism. Collectively, our designed dimeric Tat peptides with high antimicrobial activity and rapid bactericidal activity appear to be excellent candidates for future development as novel antimicrobial agents. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial activity,bactericidal mechanism and LPS‐neutralizing activity of the cell‐penetrating peptide pVEC and its analogs

pVEC is a cell‐penetrating peptide derived from the murine vascular endothelial‐cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)‐neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4–16 μM) against Gram‐positive and Gram‐negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 μM. These peptides induced a near‐complete membrane depolarization (more than 80%) at 4 μM against Staphylococcus aureus and a significant dye leakage (35–70%) from bacterial membrane‐mimicking liposome at a concentration as low as 1 μM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog‐derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor‐α release in LPS‐stimulated mouse macrophage RAW264.7 cells at 10 to 50 μM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS‐neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A β‐amino acid modified heptapeptide containing a designed recognition element disrupts fibrillization of the amyloid β‐peptide

We study the complex formation of a peptide βAβAKLVFF, previously developed by our group, with Aβ(1–42) in aqueous solution. Circular dichroism spectroscopy is used to probe the interactions between βAβAKLVFF and Aβ(1–42), and to study the secondary structure of the species in solution. Thioflavin T fluorescence spectroscopy shows that the population of fibers is higher in βAβAKLVFF/Aβ(1–42) mixtures compared to pure Aβ(1–42) solutions. TEM and cryo‐TEM demonstrate that co‐incubation of βAβAKLVFF with Aβ(1–42) causes the formation of extended dense networks of branched fibrils, very different from the straight fibrils observed for Aβ(1–42) alone. Neurotoxicity assays show that although βAβAKLVFF alters the fibrillization of Aβ(1–42), it does not decrease the neurotoxicity, which suggests that toxic oligomeric Aβ(1–42) species are still present in the βAβAKLVFF/Aβ(1–42) mixtures. Our results show that our designed peptide binds to Aβ(1–42) and changes the amyloid fibril morphology. This is shown to not necessarily translate into reduced toxicity. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.