José E. Barboza-Corona

Activity of bacteriocins synthesized by Bacillus thuringiensis against Staphylococcus aureus isolates associated to bovine mastitis

Antimicrobial therapy is a useful tool to control bovine mastitis caused by Staphylococcus aureus, as consequence an increase in staphylococci resistant cases has been registered. Alternative strategies are desirable and bacteriocins represent attractive control agents to prevent bovine mastitis. The aim of this work was to evaluate the activity of five bacteriocins synthesized by Bacillus thuringiensis against S. aureus isolates associated to bovine mastitis. Fifty S. aureus isolates were recovered from milk composite samples of 26 Holstein lactating cows from one herd during September 2007 to February 2008 in México and susceptibility of those isolates to 12 antibiotics and 5 bacteriocins from B. thuringiensis was evaluated. S. aureus isolates were mainly resistant to penicillin (92%), dicloxacillin (86%), ampicillin (74%) and erythromycin (74%); whereas susceptibility to gentamicin, trimethoprim and tetracycline was detected at, respectively, 92%, 88%, and 72%. All S. aureus isolates showed susceptibility to the five bacteriocins synthesized by B. thuringiensis, mainly to morricin 269 and kurstacin 287 followed by kenyacin 404, entomocin 420 and tolworthcin 524. Our results showed that S. aureus isolates had differences in the antimicrobial resistance patterns and were susceptible to bacteriocins produced by B. thuringiensis, which could be useful as an alternative method to control bovine mastitis.

Enhanced synthesis and antimicrobial activities of bacteriocins produced by Mexican strains of Bacillus thuringiensis

Recently, we reported the synthesis of five bacteriocin-like inhibitor substances (Bt-BLIS: morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524) by Mexican strains of Bacillus thuringiensis. Here we show that, collectively, these Bt-BLIS have a moderate to broad spectrum of antibacterial activity, being toxic to clinically significant against Gram-positive and Gram-negative bacteria, including common etiological agents of human diseases, such as strep throat and scarlet fever, septicemia, pneumonia, urinary tract infection, and emetic and gastrointestinal syndromes. Although synthesis of the five Bt-BLIS was independent of the presence of a target inducing bacterium, we demonstrated for the first time that a proteinaceous component(s) secreted by, or liberated by proteolytic cleavage of Bacillus cereus 183 following treatment with proteinase K, enhanced Bt-BLIS synthesis.

Development of a recombinant strain of Bacillus thuringiensis subsp. kurstaki HD-73 that produces the endochitinase ChiA74.

Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine–Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately 30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease in net Cry1Ac synthesis and toxicity observed with the recombinant strain.

Molecular Cloning and Purification of an Endochitinase From Serratia marcescens (Nima)

An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5αF′, and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5′-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0–10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55°C.

Molecular Detection and Sensitivity to Antibiotics and Bacteriocins of Pathogens Isolated from Bovine Mastitis in Family Dairy Herds of Central Mexico

Thirty-two farms (n = 535 cows) located in the state of Guanajuato, Mexico, were sampled. Pathogens from bovine subclinical mastitis (SCM) and clinical mastitis (CLM) were identified by 16S rDNA and the sensitivity to both antibiotics and bacteriocins of Bacillus thuringiensis was tested. Forty-six milk samples were selected for their positive California Mastitis Test (CMT) (≥3) and any abnormality in the udder or milk. The frequency of SCM and CLM was 39.1% and 9.3%, respectively. Averages for test day milk yield (MY), lactation number (LN), herd size (HS), and number of days in milk (DM) were 20.6 kg, 2.8 lactations, 16.7 animals, and 164.1 days, respectively. MY was dependent on dairy herd (DH), LN, HS, and DM (P < 0.01), and correlations between udder quarters from the CMT were around 0.49 (P < 0.01). Coagulase-negative staphylococci were mainly identified, as well as Staphylococcus aureus, Streptococcus uberis, Brevibacterium stationis, B. conglomeratum, and Staphylococcus agnetis. Bacterial isolates were resistant to penicillin, clindamycin, ampicillin, and cefotaxime. Bacteriocins synthesized by Bacillus thuringiensis inhibited the growth of multiantibiotic resistance bacteria such as S. agnetis, S. equorum, Streptococcus uberis, Brevibacterium stationis, and Brachybacterium conglomeratum, but they were not active against S. sciuri, a microorganism that showed an 84% resistance to antibiotics tested in this study.

Antimicrobial Peptides: Current and Potential Applications in Biomedical Therapies

The evolution of pathogenic bacteria has allowed many microbes to develop resistance mechanisms against conventional antibiotics, leading to the search for new therapeutic alternatives. As such, the clinical uses of antimicrobial peptides (AMPs) are among the most promising alternative options to circumvent the proliferation of antibiotic resistant pathogens. AMPs are produced by a wide variety of organisms and have a broad and largely nonspecific activity, a characteristic that strongly qualifies them as potent candidates for pharmacological applications. Indeed, the continuous discovery of new AMP groups in diverse microorganisms has expanded their potential as a new generation of antimicrobial agents for treating bacterial diseases in humans and also in animals. Intriguingly, the broad spectrum of biological activities reported for many of these molecules suggests that AMPs could also be incorporated in integrative regimen strategies against viral, fungal, and parasitic diseases and cancer, as well as in modulation of the immune system. These possibilities of uses reinforce the importance of studying the biological and applied properties of AMPs. The articles contained in the present issue include both reviews and basic scientific studies focused on characterizing AMPs from different sources to evaluate their numerous biological activities. This issue comprises the description of effects of naturally occurring AMPs from bacteria, plants, humans, and pigs as well as the effect of synthetic AMPs derived from bovines and humans. In addition, work related to the regulation of AMP expression is also included. AMPs are part of the innate response elicited by most living forms. In plants, they are produced ubiquitously in roots, seeds, flowers, stems, and leaves, highlighting their physiological importance. The contribution by C. E. Salas et al. “Biologically Active and Antimicrobial Peptides from Plants,” provides an overview of what is currently known about bioactive peptides from plants, focusing on their antimicrobial activity and their role in the plant-signaling network and offering perspectives on their potential application. The wide-ranging functionality of AMPs against infection and disease of the urinary tract expands the list of effects beyond the “antimicrobial effects” originally assigned to them. In the paper by J. Lo and D. Lange “Current and Potential Applications of Host-Defense Peptides and Proteins in Urology,” the authors discuss the existing and possible applications of these host-defense peptides in the field of urology. Transkingdom signaling is a mechanism in which molecules produced by bacteria, such as cyclodipeptides (CDPs), positively or negatively affect the development, growth, or differentiation of eukaryotic organisms. The paper by D. Vazquez-Rivera et al. “Cytotoxicity of Cyclodipeptides from Pseudomonas aeruginosa PAO1 Leads to Apoptosis in Human Cancer Cell Lines” describes that a CDP mixture promoted apoptosis in cultures of HeLa and Caco-2 cell lines in a dose-dependent manner, with 50% inhibitory concentration (IC50) of 0.53 and 0.66 mg/mL, respectively. Bacteriocins are AMPs synthesized by prokaryotes that inhibit or kill phylogenetically related and/or unrelated microorganisms that share the same microbial niche. These peptides have a potential for diversified use in the food and pharmaceutical industries, agriculture, and apiculture. In the work by M. F. Leon-Galvan et al. entitled “Molecular Detection and Sensitivity to Antibiotics and Bacteriocins of Pathogens Isolated from Bovine Mastitis in Family Dairy Herds of Central Mexico,” the authors showed that bacteriocins synthesized by Bacillus thuringiensis, a Gram-positive bacterium, inhibited the growth of multiantibiotic resistance bacteria responsible of mastitis, a very important disease in bovines. The anticancer activity elicited by AMPs has stimulated intriguing prospects for their use in chemotherapy as cancer remains a cause of high morbidity and mortality worldwide of utmost interest. J. Guzman-Rodriguez et al. in the paper entitled “Plant Antimicrobial Peptides as Potentials Anticancer Agents” provide an overview of plant AMPs (thionins, defensins, and cyclotides) with anticancer activities with particular emphasis on their mode of action, their selectivity, and their efficacy. Innate immunity defense is upregulated by antimicrobial peptide elicitors, which are defined as physical, chemical, and biological agents that promote upregulation of endogenous AMPs. The paper by L. A. C. Diaz et al. entitled “Ascorbic Acid, Ultraviolet C Rays, and Glucose but Not Hyperthermia Are Elicitors of Human β-Defensin 1 mRNA in Normal Keratinocytes” investigates the effects of hyperthermia, ultraviolet A rays, and ultraviolet C rays, as well as glucose and ascorbic acid, on the regulation of human β-defensin 1 (DEFB1), cathelicidin (CAMP), and interferon-γ (IFNG) genes in normal human keratinocytes. The effects of AMPs on bacteria have been extensively studied. However, fewer reports exist regarding their effects on protozoa. The work by J. L. Hernandez-Flores entitled “Effect of Recombinant Prophenin 2 on the Integrity and Viability of Trichomonas vaginalis” reports that the propeptide and the processed peptide of prophenin 2 affect the integrity and growth of T. vaginalis and that proprophenin displays some resistance to proteolysis by T. vaginalis proteinases. Its effect on T. vaginalis, as well as its low hemolytic activity and short-time stability to parasite proteinases, makes prophenin an interesting candidate for synergistic or alternative treatment against T. vaginalis. The development of novel synthetic analogs of AMPs could enhance their activities, facilitating the development of new drugs. In their paper “Antibacterial Activity of Synthetic Peptides Derived from Lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212”, M. A. Leon et al. demonstrate that peptides derived from human and bovine lactoferricin exhibit higher or similar activity against E. coli (MIC 4–33 μM) and E. faecalis (MIC 10–33 μM) compared with lactoferricin protein. Their work shows that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity. The low amount of bacteriocins obtained from direct purification from natural producers and the elevated production costs of chemical synthesis have stimulated interests in producing these proteins in heterologous microbial hosts through recombinant genetic manipulations. In the paper by S. Arbulu et al. entitled “Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris”, the authors evaluated the cloning and functional expression and production of several broad spectrum bacteriocins in recombinant Pichia pastoris and assayed these antimicrobials against Listeria monocytogenes CECT4032, E. coli O157:H7, Yersinia ruckeri LMG3279, Campylobacter jejuni ATCC33560, and C. jejuni NCTC11168. We hope that this special issue would shed light on major developments in the area of AMPs and attract attention by the scientific community to pursue further investigations leading to the rapid implementation of these molecules in chemotherapeutics.

Isolation of a new Mexican strain of Bacillus subtilis with antifungal and antibacterial activities.

Although several strains of B. subtilis with antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a native B. subtilis strain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis 21) demonstrated in vitro antagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected with Rhizoctonia solani and Fusarium verticillioides and treated with B. subtilis 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants.

Generation of chitin-derived oligosaccharides toxic to pathogenic bacteria using ChiA74, an endochitinase native to Bacillus thuringiensis

Aims:  To demonstrate that an endochitinase (ChiA74) native to Bacillus thuringiensis can be used to generate chitin‐derived oligosaccharides (OGS) with antibacterial activity against a number of aetiological agents of disease, including bacteria that cause diarrhoeal and emetic syndromes in humans.

Bacillus thuringiensis subsp. israelensis producing endochitinase ChiA74Δsp inclusions and its improved activity against Aedes aegypti.

The objective of this study was to produce stable inclusions of chitinase ChiA74Δsp in Bacillus thuringiensis subsp. israelensis (Bti) and to assay its insecticidal activity against Aedes aegypti larvae.

The endochitinase ChiA Btt of Bacillus thuringiensis subsp. tenebrionis DSM-2803 and its potential use to control the phytopathogen Colletotrichum gloeosporioides.

Bacillus thuringiensis subsp. tenebrionis DSM‐2803 has been studied extensively and spore/crystal mixtures of this strain are used widely in commercial products to control coleopteran pests. The endochitinase chiA Btt gene of B. thuringiensis subsp. tenebrionis DSM‐2803 was cloned and expressed in Escherichia coli. The recombinant 6x‐histidine tagged protein (rChiA Btt, ~74 kDa), was purified by a HiTrap Ni affinity column. The Km of rChiA Btt was 0.847 μmol L−1 and its optimal activity occurred at pH 7 and ~40°C. Most divalent cations reduced endochitinase activity but only Hg+2 abolished activity of the enzyme. We report for the first time the characterization of a chitinase synthesized by B. thuringiensis subsp. tenebrionis DSM‐2803, and show that the purified rChiA74 Btt reduced the radial growth and increased the hyphal density of Colletotrichium gloeosporioides, the etiological agent of “anthracnose” in plants.