Microcin PDI Inhibits Antibiotic-Resistant Strains of Escherichia coli and Shigella through a Mechanism of Membrane Disruption and Protection by Homotrimer Self-Immunity.

Microcin PDI (MccPDI), a class IIa microcin that is produced by Escherichia coli strains 25 and 284, is known to inhibit foodborne pathogenic enterohemorrhagic E. coli serotypes O157:H7 and O26. Here we demonstrate that MccPDI can inhibit Shigella strains and E. coli isolates that are multidrug resistant, the latter including strains known to cause urinary tract infections in people and companion animals. Two exceptions out of 17 strains were identified. One of the two resistant E. coli isolates (AR0349) has a mutation in a critical amino acid residue that was identified in previous work as a requisite for the MccPDI precursor protein (McpM) to interact with outer membrane porin F (OmpF) on susceptible cells. The second resistant E. coli strain (MAD 96) had no mutations in ompF, but it was PCR positive for two antimicrobial peptides, of which colicin Ia/Ib likely inhibits the MccPDI-producing strain during coculture. Recombinant McpM was still effective against strain MAD 96. In an assessment of how MccPDI affects susceptible strains, results from both an extracellular ATP assay and a nucleic acid staining assay were consistent with membrane damage, while the addition of 200- to 600-Da polyethylene glycol (PEG) to cocultures protected against MccPDI (>600-Da PEG did not provide protection). Further studies using a paraformaldehyde cross-linking experiment and a bacterial two-hybrid assay demonstrated that MccPDI immunity protein (McpI) forms a multimeric complex with itself and presumably protects the producer strain from within the periplasm through an unknown mechanism.IMPORTANCE Microcins represent potential alternatives to conventional antibiotics for human and veterinary medicine. For them to be applied in this manner, however, we need to better understand their spectrum of activity, how these proteins interact with susceptible cells, and how producer cells are protected against the antimicrobial properties of the microcins. For microcin PDI (MccPDI), we report that the spectrum of activity likely includes most E. coli strains due to a conserved binding motif found on an outer membrane protein. Shigella has this motif as well and is susceptible to MccPDI killing via damage to the bacterial membrane. Receptor specificity suggests that these proteins could be used without causing large-scale disruptions to a microbiota, but this also increases the likelihood that resistance can evolve via random mutations. As with conventional antibiotics, good stewardship will be needed to preserve the efficacy of microcins should they be deployed for clinical use.

The glycocins: in a class of their own.

First reported in 2011, glycocins (glycosylated bacteriocins) are bacterial toxins that constitute a subset of ribosomally synthesised and post-translationally modified peptide (RiPP) natural products. Three NMR structures (glycocin F, ASM1 and sublancin 168), two with helix-loop-helix Cs α/α folds, are deposited in the PDB. Each structure contains a monosaccharide ß-S-linked to a cysteine side chain. Three more glycocins (thurandacin, and enterocins F4-9 and 96) have been biochemically characterised, and others predicted on the basis of bioinformatic analyses. Only glycocin F, ASM1 and enterocin F4-9 are unequivocally glycoactive. This review probes the structure-function relationships of four types of nested disulfide-bonded glycocins.

A large scale prediction of bacteriocin gene blocks suggests a wide functional spectrum for bacteriocins.

BACKGROUND: Bacteriocins are peptide-derived molecules produced by bacteria, whose recently-discovered functions include virulence factors and signaling molecules as well as their better known roles as antibiotics. To date, close to five hundred bacteriocins have been identified and classified. Recent discoveries have shown that bacteriocins are highly diverse and widely distributed among bacterial species. Given the heterogeneity of bacteriocin compounds, many tools struggle with identifying novel bacteriocins due to their vast sequence and structural diversity. Many bacteriocins undergo post-translational processing or modifications necessary for the biosynthesis of the final mature form. Enzymatic modification of bacteriocins as well as their export is achieved by proteins whose genes are often located in a discrete gene cluster proximal to the bacteriocin precursor gene, referred to as context genes in this study. Although bacteriocins themselves are structurally diverse, context genes have been shown to be largely conserved across unrelated species. METHODS: Using this knowledge, we set out to identify new candidates for context genes which may clarify how bacteriocins are synthesized, and identify new candidates for bacteriocins that bear no sequence similarity to known toxins. To achieve these goals, we have developed a software tool, Bacteriocin Operon and gene block Associator (BOA) that can identify homologous bacteriocin associated gene blocks and predict novel ones. BOA generates profile Hidden Markov Models from the clusters of bacteriocin context genes, and uses them to identify novel bacteriocin gene blocks and operons. RESULTS AND CONCLUSIONS: We provide a novel dataset of predicted bacteriocins and context genes. We also discover that several phyla have a strong preference for bacteriocin genes, suggesting distinct functions for this group of molecules. SOFTWARE AVAILABILITY: https://github.com/idoerg/BOA.

Properties of Bac W42, a bacteriocin produced by Bacillus subtilis W42 isolated from Cheonggukjang.

Ten Bacillus strains with antimicrobial activities were isolated from Cheonggukjang produced at different parts in Korea. They all inhibited Listeria monocytogenes ATCC 19111 and nine inhibited Bacillus cereus ATCC 14579. Four isolates (W42, H27, SKE 12, and K21) showing strong inhibiting activities were identified as B. subtilis. B. subtilis W42 was the most inhibiting strain. The antimicrobial activity of culture supernatant from B. subtilis W42 was destroyed completely by proteinase K treatment, indicating that a bacteriocin was the responsible agent. The bacteriocin, Bac W42, was most stable at pH 7 and stable between pH 3-6 and 8-9. Bac W42 was stable up to 80°C. BHI (brain heart infusion) and TSB (tryptic soy broth) were the best media for the activity (320 AU/ml) followed by LB (160 AU/ml). Bac W42 was partially purified by column chromatographies. The specific activity was increased from 1,151.2 AU/ml to 9,043.5 AU/ml and the final yield was 26.3%. Bac W42 was 5.4 kDa in size as determined by SDS-PAGE. Bac W42 showed bactericidal activity against L. monocytogenes ATCC 19111.

Periodontal pathogens interfere with quorum-sensing-dependent virulence properties in Streptococcus mutans.

BACKGROUND AND OBJECTIVE: The mechanism by which periodontal pathogens dominate at disease sites is not yet understood. One possibility is that these late colonizers antagonize the quorum-sensing systems of early colonizers and render those early colonizers less resistant to environmental factors. In this study, we utilized Streptococcus mutans, a well-documented oral Streptococcus with many quorum-sensing-dependent properties, as an example of an earlier colonizer antagonized by periodontal pathogens. MATERIAL AND METHODS: In this study, S. mutans NG8 and S. mutans LT11 were used in experiments assessing transformation, and S. mutans BM71 was used in experiments investigating bacteriocin production. The effects of the periodontal pathogens Porphyromonas gingivalis and Treponema denticola on these competence-stimulating peptide-dependent properties were evaluated in mixed-broth assays. RESULTS: Both P. gingivalis (either live bacteria or membrane vesicles) and T. denticola antagonized transformation in S. mutans NG8 and LT11. The production of bacteriocin by S. mutans BM71 was also inhibited by P. gingivalis and T. denticola. Boiling of these late colonizers before addition to the broth cultures abolished their ability to inhibit S. mutans transformation and bacteriocin production. P. gingivalis and T. denticola inactivated S. mutans exogenous competence-stimulating peptide, whereas the boiled bacteria did not. CONCLUSIONS: This study demonstrated that periodontal pathogens antagonize S. mutans quorum-sensing properties. This may render S. mutans less virulent and less resistant to environmental antibacterial factors.

Identification of DysI, the immunity factor of the streptococcal bacteriocin dysgalacticin.

DysI is identified as the protein that confers specific immunity to dysgalacticin, a plasmid-encoded streptococcal bacteriocin. dysI is transcribed as part of the copG-repB-dysI replication-associated operon. DysI appears to function at the membrane level to prevent the inhibitory effects of dysgalacticin on glucose transport, membrane integrity, and intracellular ATP content.

Kinetic modeling of the thermal inactivation of bacteriocin-like inhibitory substance p34.

Optimization of thermal processes relies on adequate degradation kinetic models to warrant food safety and quality. The knowledge on thermal inactivation kinetics of antimicrobial peptides is necessary to allow for their adequate use as natural biopreservatives in the food industry. In this work, thermal inactivation of the previously characterized bacteriocin-like inhibitory substance (BLIS) P34 was kinetically investigated within the temperature range of 90-120 degrees C. Listeria monocytogenes ATCC 7644 was used as the indicator microorganism for antimicrobial activity. Applicability of various inactivation models available in the literature was critically evaluated. The first-order model provided the best description of the kinetics of inactivation over the selected temperatures, with k values between 0.059 and 0.010 min(-1). D and k values decreased and increased, respectively, with increasing temperature, indicating a faster inactivation at higher temperatures. Results suggest that BLIS P34 is thermostable, with a z value of 37.74 degrees C and E(a) of 72 kJ mol(-1).

Pesquisa de substâncias antagonistas tipo bacteriocinas produzidas por amostras de Staphylococcus aureus meticilina resistentes isoladas de pacientes inter-nados no Hospital Regional do Município de Barbacena, Minas Gerais / Research of antagonist substances bacteriocins produced by samples of methicillin-resistant staphylococcus aureus isolated from patients admitted to Regional Hospital of Barbacena-City, Minas Gerais

Objetivos: detectar a produção de bacteriocinas, determinar a suscetibilidade das amostras coletadas a essas substãncias e estabelecer possível correlação entre bacteriocinas e sua ação antimicrobiana. Métodos: foram coletadas 14 amostras isoladas de processos infecciosos causados por cepas de Staphylococcus aureus/Meticilina resistentes (MRSA),de pacientes do município de Barbacena-MG. Essas amostras foram previamente identificadas, caracterizadas com o MRSA , preservadas à temperatura de -80ºC em freezer, em meio apropriado de manutenção com crioprotetores, skim-milke, e submetidas a testes no laboratório. Resultados: foram realizados 196 testes, sendo que 100 por cento das amostras testadas mostraram -se produtoras de bacteriocinas. As cepas MRSAs eram, em 71,4 por cento dos casos, sensíveis à ação de bacteriocinas produzidas pelas bactérias testadas, tendo o seu crescimento inibido. O fenômeno de autoantagonismo foi também observado em 57,1 por cento das bactérias estudadas. Conclusão: os resultados demonstraram o alto potencial antimicrobiano das MRSAs isoladas. O autoantagonismo exerce marcante papel na manutenção do equilíbrio entre as com unidades microbianas que constituem a flora humana normal. Diante do aumento e da rápida disseminação de bactérias patógenas multirresistentes, encontradas em ambientes hospitalares e na comunidade, as bacteriocinas estudadas poderiam oferecer solução alternativa na terapêutica de infecções envolvendo esses patógenos.

Bacteriocin production by Pediococcus pentosaceus isolated from marula (Scerocarya birrea).

Strain ST44AM, isolated from marula, was identified as Pediococcus pentosaceus based on biochemical tests, sugar fermentation reactions (API 50CHL), PCR with species-specific primers and 16S rDNA sequencing. Strain ST44AM produces a 6.5 kDa class IIa bacteriocin, active against lactic acid bacteria, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria innocua, Listeria ivanovii subsp. ivanovii and Listeria monocytogenes. The peptide is inactivated by proteolytic enzymes, but not when treated with alpha-amylase, Triton X-100, Triton X-114, SDS, Tween 20, Tween 80, urea, NaCl and EDTA. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 degrees C for 120 min or 121 degrees C for 20 min. The mode of activity against L. ivanovii susbp. ivanovii ATCC19119 and Enterococcus faecium HKLHS is bactericidal, resulting in cell lyses and enzyme- and DNA-leakage. No significant differences in cell growth and bacteriocin production were observed when strain ST44AM was cultured in MRS broth at 26 degrees C, 30 degrees C and 37 degrees C for 24 h and tested against the same target strain. L. ivanovii subsp. ivanovii ATCC 19119 and E. faecium HKLHS did, however, differ in sensitivity to bacteriocin ST44AM (3.3x10(6) AU/mL and 2.6x10(4) AU/mL, respectively). Peptide ST44AM adsorbs at high levels (1600 AU/mL) to producer cells. Bacteriocin ST44AM may be a derivative of pediocin PA-1. This is the first report on the presence of P. pentosaceus in marula and a pediocin PA-1 derivative produced by this species. We are also the first to report on the synergetic effect ciprofloxacin has on a pediocin-like bacteriocin.

Plasmid pAMS1-encoded, bacteriocin-related "Siblicide" in Enterococcus faecalis.

The Enterococcus faecalis class IIa bacteriocin MC4-1 encoded by the sex pheromone-responding, multiple-antibiotic resistance plasmid pAMS1 exhibits "siblicidal" (sibling-killing) activity under certain conditions. Stabs of plasmid-containing cells on solid medium containing lawns of bacteria of the same (plasmid-containing) strain give rise to zones of inhibition. If the plasmid-containing host also produces gelatinase, bacteriocin cannot be detected.

DNA sequencing and homologous expression of a small peptide conferring immunity to gassericin A, a circular bacteriocin produced by Lactobacillus gasseri LA39.

Gassericin A, produced by Lactobacillus gasseri LA39, is a hydrophobic circular bacteriocin. The DNA region surrounding the gassericin A structural gene, gaaA, was sequenced, and seven open reading frames (ORFs) of 3.5 kbp (gaaBCADITE) were found with possible functions in gassericin A production, secretion, and immunity. The deduced products of the five consecutive ORFs gaaADITE have homology to those of genes involved in butyrivibriocin AR10 production, although the genetic arrangements are different in the two circular bacteriocin genes. GaaI is a small, positively charged hydrophobic peptide of 53 amino acids containing a putative transmembrane segment. Heterologous expression and homologous expression of GaaI in Lactococcus lactis subsp. cremoris MG1363 and L. gasseri JCM1131(T), respectively, were studied. GaaI-expressing strains exhibited at least sevenfold-higher resistance to gassericin A than corresponding control strains, indicating that gaaI encodes an immunity peptide for gassericin A. Comparison of GaaI to peptides with similar characteristics found in the circular bacteriocin gene loci is discussed.

Nuclear magnetic resonance solution structure of PisI, a group B immunity protein that provides protection against the type IIa bacteriocin piscicolin 126, PisA.

Lactic acid bacteria produce and secrete bacteriocins. These bacteriocins are potent antimicrobial peptides that are active against other closely related bacteria. As a means of self-protection, producer organisms also express immunity proteins. Immunity proteins are generally located on the same genetic locus and are cotranscribed with the bacteriocin. Although some cross immunity between bacteriocins has been observed, immunity proteins are typically highly specific. Immunity proteins for the type IIa bacteriocins range from 81 to 115 amino acids in length and display substantial variation in their sequences. Nonetheless, such immunity proteins have been classified into three groupings (groups A, B, and C) according to sequence homology. The structures of a group C (ImB2) and two group A (EntA-im and PedB) immunity proteins have previously been reported. We herein report the nuclear magnetic resonance solution structure of the remaining class of the type IIa immunity proteins. PisI, a 98-amino acid protein, is a group B immunity protein conferring immunity against piscicolin 126 (PisA). Like ImB2, EntA-im, and PedB, PisI folds into a globular protein in aqueous solution and contains an antiparallel four-helix bundle. Compared to ImB2 and EntA-im, PisI has a substantially longer and more flexible N-terminus, but a shorter C-terminus. No direct interaction between the bacteriocin and immunity protein is observed by NMR in either aqueous or membrane mimicking environments. This further suggests that the mechanism that mediates immunity is not due to a direct bacteriocin-immunity protein interaction but rather is receptor-mediated. It has now been confirmed that the four-helix bundle is indeed a structural motif among the type IIa immunity proteins.

Purification and partial characterization of novel bacteriocin L23 produced by Lactobacillus fermentum L23.

Lactobacillus fermentum strain L23 produced a small bacteriocin, designated bacteriocin L23, with an estimated molecular mass of < 7000 Da. Isolation, purification, and partial characterization of bacteriocin L23 are described. It displayed a wide inhibitory spectrum including both Gram-negative and Gram-positive pathogenic strains and two species of Candida. The antibacterial activity of cell-free culture supernatant fluid was not affected by catalase or urease but was abolished by the proteolytic enzymes trypsin and protease VI. Bacteriocin L23 was heat stable (60 min at 100 degrees C) and showed inhibitory activity over a wide pH range (4.0 to 7.0). The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin sample was prepared by a process of ammonium sulfate precipitation, gel filtration, thin-layer chromatography, bioautography, and reversed-phase HPLC.

Influence of baking enzymes on antimicrobial activity of five bacteriocin-like inhibitory substances produced by lactic acid bacteria isolated from Lithuanian sourdoughs.

AIM: To evaluate the effect of four different baking enzymes on the inhibitory activity of five bacteriocin-like inhibitory substances (BLIS) produced by lactic acid bacteria (LAB) isolated from Lithuanian sourdoughs. METHODS AND RESULTS: The overlay assay and the Bioscreen methods revealed that the five BLIS exhibited an inhibitory effect against spore germination and vegetative outgrowth of Bacillus subtilis, the predominant species causing ropiness in bread. The possibility that the observed antibacterial activity of BLIS might be lost after treatment with enzymes used for baking purposes was also examined. CONCLUSIONS: The enzymes tested; hemicellulase, lipase, amyloglucosidase and amylase had little or no effect on the majority of the antimicrobial activities associated with the five BLIS studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests a potential application in the sourdough baking industry for these antimicrobial producing LAB strains in the control of B. subtilis spore germination and vegetative outgrowth.

Bacteriocin-like inhibitory substances from Campylobacter spp.

Twenty-five Campylobacter isolates were screened for production of antimicrobial substances using a deferred antagonism assay. Sixteen isolates showed activity against either Staphylococcus aureus, Salmonella enterica serovar Enteritidis or Candida albicans. The inhibitory activity was sensitive to treatment with pronase E, trypsin and pepsin, suggesting that the antimicrobial compound(s) are proteinaceous. Activity spectra of isolates included S. aureus, Micrococcus luteus, Streptococcus sp., Bacillus subtilis, a drug-resistant clinical isolate of S. aureus and one isolate of C. albicans. Producing isolates showed cross-immunity and inhibitory activity was only observed on solid media. The findings of this study suggest that Campylobacter produces proteinaceous inhibitory substances.

NMR solution structure of the precursor for carnobacteriocin B2, an antimicrobial peptide from Carnobacterium piscicola.

Type IIa bacteriocins, which are isolated from lactic acid bacteria that are useful for food preservation, are potent antimicrobial peptides with considerable potential as therapeutic agents for gastrointestinal infections in mammals. They are ribosomally synthesized as precursors with an N-terminal leader, typically 18-24 amino acid residues in length, which is cleaved during export from the producing cell. We have chemically synthesized the full precursor of carnobacteriocin B2, precarnobacteriocin (preCbnB2), which has a C-terminal amide rather than a carboxyl, and also produced preCbnB2(1-64), which is missing two amino acid residues at the C-terminus (Arg65 and Pro66), via expression in Escherichia coli as a maltose-binding protein fusion that is then cut with Factor Xa. PreCbnB2(1-64) is readily labeled with (15)N and (13)C for NMR studies using the latter approach. Multidimensional NMR analysis of preCbnB2(1-64) shows that, like the parent bacteriocin, it exists as a random coil in water but assumes a defined conformation in water/trifluoroethanol mixtures. In 70 : 30 trifluoroethanol/water, the 3D structure of the preCbnB2 section corresponding to the mature bacteriocin is essentially the same as reported previously by us for carnobacteriocin B2 (CbnB2). This structure maintains the highly conserved alpha-helix corresponding to residues 20-38 of CbnB2 that is believed to be responsible for interaction with a target receptor in sensitive cells, including Listeria monocytogenes. PreCbnB2 also has a second alpha-helix from residues 3-13 (i.e. -15 to -5 relative to CbnB2) in the leader section of the peptide. This helix appears to be conserved in related type IIa bacteriocin precursors based on sequence analysis. It is likely to be a key recognition element for export and processing, and is probably responsible for the considerably reduced antimicrobial activity of preCbnB2. The latter effect may assist the producing cell in avoiding the toxic effects of the bacteriocin. This is the first 3D structure determined for a prebacteriocin from lactic acid bacteria.

Inhibition of enterocin CRL35 antibiotic activity by mono- and divalent ions.

AIM: The aim of this work was to study the influence of different cations on the enterocin CRL35 activity. METHODS AND RESULTS: The antilisterial activity of enterocin CRL35 was tested by performing viability curves and measuring the dissipation of the proton motive force by fluorescent methods upon the addition of Ca2+, Mg2+, Li+, K+ and Na+ chlorides. The peptide uptake by sensitive cells was studied in the different conditions as well. The addition of calcium and magnesium chlorides (0.5-2 mmol l(-1)) induced an inhibition of the peptide activity. Potassium, sodium and lithium chlorides have an inhibitory effect as well, but at different range of concentration compared with divalent cations (50-150 mmol l(-1)). Interestingly, we found a differential protection effect among monovalent ions, KCl being almost nonprotective, meanwhile LiCl shows the stronger effect and NaCl has an intermediate effect. The ion effect depends on the pH, being more efficient in acidic media. Both mono and divalent ions inhibited the ability of the peptide to dissipate the transmembrane electric potential and pH gradient. Furthermore, the peptide uptake was also inhibited. CONCLUSIONS: The enterocin CRL35 activity is strongly dependent on the pH and the nature of the salts present in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will allow definition of the best system in which this peptide can be applied as biopreservative.

Considerations relating to the epidemiology of human immunodeficiency virus infection: the impact of bacterial antigens and consequences for treatment.

OBJECTIVE: A treatment for patients with human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) is presented, which is based on an isopathic method that appears to be effective in eliminating bacterial antigens from the body. The concept is based on a new hypothesis concerning the outbreak and spread of AIDS in Africa and worldwide. SUBJECTS AND DESIGN: Laboratory data are presented from five European and seven African patients with HIV. RESULTS: Oral administration of ultra-low doses of a lysate of Staphylococcus aureus Cowan I (12c potency) resulted in a significant increase of CD4 T-cell subsets and CD4/CD8 ratios in patients with HIV infection as well as in advanced stages of HIV disease, concomitant with the improvement of clinical HIV-related symptoms. CONCLUSIONS: Based on epidemiologic data, the beginning of the African AIDS epidemic is related-to time, place, and circumstances-to the initial large-scale introduction of antibiotics in areas of Central Africa that would later comprise the AIDS belt. It is concluded that certain antimicrobial agents can enhance the formation of persistent bacterial superantigens, which may indicate a link between asymptomatic HIV carriers and the development of AIDS. According to this view, superantigens and bacterial cell wall components remaining in the body after antibiotic treatment cause a permanent activation of the immune system and would thus favor T-cell infection and viral replication in HIV-infected individuals.

H(2)O(2) produced by viridans group streptococci may contribute to inhibition of methicillin-resistant Staphylococcus aureus colonization of oral cavities in newborns.

In an accompanying report, we showed that viridans group streptococci may prevent methicillin-resistant Staphylococcus aureus (MRSA) colonization of the oral cavities of newborns. In the present study, we investigated the mechanism of prevention in vitro. Most viridans group streptococci had bacteriocin-like activity and killed MRSA, Burkholderia cepacia, Enterobacter aerogenes, and Pseudomonas aeruginosa; however, Escherichia coli, Enterobacter cloacae, and Candida albicans were resistant. The activity was induced only by H(2)O(2)-secreting strains and was inhibited by horseradish peroxidase or catalase in a dose-dependent manner. The mean concentration of H(2)O(2) produced by 18 strains of viridans group streptococci (1 x 10(8) cfu in 200 microL of culture medium+/-standard deviation was 1.24+/-0.60 mmol. Viridans group streptococci inhibited MRSA growth in saliva as well as in culture media. These results indicate that H(2)O(2) produced by viridans group streptococci may inhibit MRSA colonization of oral cavities in newborns.