[Show abstract][Hide abstract] ABSTRACT: Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and sulphonamides dfrA17-aadA5 and dfrA12-orfF-aadA2. One isolate ETEC_Ef-6 was found to be a multidrug-resistant (MDR) pathogen that carried both the beta-lactamase gene and class 1 integron. These data suggest the circulation of ETEC in Russia. Further investigations are necessary to study the spread of the revealed ETEC sequence types (STs) and serotypes. Their role in the etiology of diarrhea should be also estimated.
PLoS ONE 04/2015; 10(4):e0123357. DOI:10.1371/journal.pone.0123357 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lactobacillus salivarius 1077 (NRRL B-50053) was isolated from poultry intestinal materials, and in vitro anti-Campylobacter jejuni activity was demonstrated. The isolate was then used for bacteriocin production and its enrichment. The protein content of
the cell-free supernatant from the spent medium was precipitated by ammonium sulfate and dialyzed to produce the crude antimicrobial
preparation. A typical bacteriocin-like response of sensitivity to proteolytic enzymes and resistance to lysozyme, lipase,
and 100°C was observed with this preparation. The polypeptide was further purified by gel filtration, ion-exchange, and hydrophobic-interaction
chromatography. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), Edman degradation,
and isoelectrofocusing were used to characterize its 3,454-Da molecular mass, the amino acid sequence of its 37 residue components,
and the isoelectric point of pI 9.1 of the bacteriocin. Bacteriocin L-1077 contained the class IIa bacteriocin signature N-terminal
sequence YGNGV. MICs of bacteriocin L-1077 against 33 bacterial isolates (both Gram negative and Gram positive) ranged from
0.09 to 1.5 μg/ml. Subsequently, the therapeutic benefit of bacteriocin L-1077 was demonstrated in market-age (40- to 43-day-old)
broiler chickens colonized with both C. jejuni and Salmonella enterica serovar Enteritidis. Compared with untreated control birds, both C. jejuni and S. Enteritidis counts in colonized ceca were diminished by >4 log10 and S. Enteritidis counts in both the liver and the spleen of treated birds were reduced by 6 to 8 log10/g compared with those in the nontreated control birds. Bacteriocin L-1077 appears to hold promise in controlling C. jejuni/S. Enteritidis among commercial broiler chickens.
[Show abstract][Hide abstract] ABSTRACT: The therapeutic efficacy of enterocin S760, a broad spectrum antimicrobial peptide produced by Enterococcus faecium LWP760 was tested on mice infected with Bacillus anthracis M-71 to induce anthrax (second Tsenkovsky's vaccine). Intraperitoneal four-, two- or one-fold administration of the peptide in a dose of 25 mg/kg for 10 days for prophylactic (1 hour after the contamination) and therapeutic (24 hours after the contamination) purposes prevented or cured the infection in 90-100% of the mice versus the 100-percent lethality in the control (untreated animals). The antimicrobial activity of enterocin S760 against B. anthracis M-71 in vivo correlated with activity in vitro. Enterocin S760 is considered a novel promising antimicrobial for the treatment of grampositive and gramnegative infections.
Antibiotiki i khimioterapii͡a = Antibiotics and chemoterapy [sic] / Ministerstvo meditsinskoĭ i mikrobiologicheskoĭ promyshlennosti SSSR 01/2011; 56(9-10):13-8.
[Show abstract][Hide abstract] ABSTRACT: Novel bacteriocins and/or the novel lactic acid-producing strains are used for at least reducing the levels of colonization by at least one target bacteria in animals, especially poultry.
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial activity of bacteriocin S760 (enterocin) produced by Enterococcusfaecium strain LWP760 was studied. Bacteriocin S760 is a cationic, hydrophobic, and heat stable peptide with the molecular weight of 5.5 kDa and pl of 9.8. Enterocin S760 is shown to inhibit in vitro the growth both of sensitive and resistant to antibacterials gramnegative and grampositive bacteria of 25 species. MICs of the bacteriocin S760 vary between 0.05-1.6 mg/l for Escherichia coli 0157:H117, Salmonella typhimurium, Salmonella enteritidis, Campylobacter jejuni, Yersinia enterocolitica, Yersinia pseudotuberculosis, Listeria monocytogenes and Clostridium perfringens, that are main food-borne pathogens, and from 0.4-1.6 mg/l for Streptococcus pyogenes, Streptococcus pneumoniae and Corynebacterium diphteriae. It is also active against antibioticresistant strains of Staphylococcus aureus, Enterobacter cloacae, Acinetobacter baumannii (with MICs of 0.05-3 mg/l), Klebsiella pneumoniae (with MICs of 6 mg/l), Pseudomonas aeruginosa (with MICs of 0.4-25 mg/1), as well against fungi belonging to species of Candida albicans, Candida krusei and Aspergillus niger (with MICs of 0.1-0.2 mg/l). Enterocin S760 is a novel antimicrobial agents useful in medicine, veterinary and food industry.
Antibiotiki i khimioterapii͡a = Antibiotics and chemoterapy [sic] / Ministerstvo meditsinskoĭ i mikrobiologicheskoĭ promyshlennosti SSSR 01/2011; 56(1-2):3-9.
[Show abstract][Hide abstract] ABSTRACT: Bacteriocins (BCN) are antimicrobial peptides that provide potential to control bacterial infections in a variety of applications. We previously reported on three class IIa BCN molecules produced by Lactobacillus salivarius B-30514 (OR-7), Enterococcus faecium B-30746 (E 50-52), and Enterococcus durans/faecium/hirae B-30745 (E-760). These BCN are notably effective against a wide array of pathogenic bacteria. To commercially apply such BCN, adequate quantities must be produced and harvested. We determined that a combination of host producer synthesized signal peptides (SP) in the presence of both producer and inducer bacteria grown in a dilute fermentation medium enabled marked increases in the synthesis of BCN. These SP contained 24-30 amino acid residues with sizes ranging from 2095 to 3065 Da having the unique terminal carboxyl sequence of VKGLT. The inducer bacterial isolates used were Lactobacillus acidophilus B-30510 and Lactobacillus crispatus B-30884. We used a nutrient-limited medium of 10% brucella broth (containing 0.01% glucose) to enhance BCN production. Using the combination of these above three parameters enabled us to reproducibly harvest at least 200 mg of BCN/L of the spent fermentation broth. This information can be used to assist in the production of BCN.
Journal of Agricultural and Food Chemistry 05/2010; 58(10):6033-8. DOI:10.1021/jf902802z · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To demonstrate treatment efficacy of bacteriocin S760 synthesized by Enterococcus faecium 760 for septic Salmonella infection in mice.
One hundred mice, which were intraperitoneally inoculated with 100 LD50 of Salmonella enteritidis strain 92 Rif(r), received bacteriocin 1 hour (prevention) or 48 hours (treatment) after inoculation in doses 25, 50, or 100 mg/kg every 6 hours during 5 or 10 days.
Use of peptide S760 for prophylaxis in dose 50 mg/kg during 10 days prevented lethal infection in 100% of animals, whereas its use for treatment cured 70% of animals with generalized salmonellosis. Shortening of treatment course from 10 to 5 days and reducing dose of bacteriocin led to less pronounced treatment effect but in all animals it was expressed by increase of mean length of life compared to control (not treated).
Obtained results demonstrated high treatment efficacy of bacteriocin S760 during septic salmonellosis and perspectives of its use in medicine and animal health.
Zhurnal mikrobiologii, epidemiologii, i immunobiologii 01/2010;
[Show abstract][Hide abstract] ABSTRACT: The antimicrobial spectra of previously published bacteriocin E50–52 (39 a.a.; 3,932Da; pI=8.5) and bacteriocin B602
(29 a.a.; 3,864Da; pI=7.2) were determined. Named peptides were related to class IIa (pediocin-like) bacteriocins. Minimal
inhibitory concentrations (MICs) of bacteriocins have been determined for bacterial isolates that were causative agents of
nosocomial infections collected from Russian hospitals in 2003–2007, namely methicillin-resistant Staphylococcus aureus (MRSA) (n=10); Acinetobacter baumannii (n=11); Citrobacter freundii (n=8); Escherichia coli (n=9); Klebsiella pneumoniae (n=10); Proteus spp. (n=6); and Pseudomonas aeruginosa (n=10). The majority of these tested isolates have been shown to be multidrug resistant and carry genetic determinants of
antimicrobial resistance that were detected using polymerase chain reaction (PCR). The MICs of bacteriocin B602 ranged from
≤0.025–1.56μg/ml, and for bacteriocin E50–52 from 0.05 to 6.25μg/ml for all of 64 bacterial clinical isolates tested. Interestingly,
the bacteriocins studied demonstrate activity on both Gram-positive and Gram-negative bacteria. Bacteriocins E50–52 and B602
show good activity against nosocomial bacterial agents resistant to many classes of modern antibacterials used in clinical
practice. These bacteriocins should be examined as an alternative in treating infections caused by such agents.
Probiotics and Antimicrobial Proteins 12/2009; 1(2):136-142. DOI:10.1007/s12602-009-9027-6
[Show abstract][Hide abstract] ABSTRACT: Broiler chickens are widely considered an important source of human exposure to Campylobacter jejuni because of the high numbers found colonizing the chicken gut and the consequent contamination of processed carcasses. We hoped to intervene in C. jejuni gut colonization by using a defined probiotic. Chicken intestinal contents were screened for diverse bacterial isolates that manifested C. jejuni inhibition. These antagonistic bacteria were fed directly to chickens before or after C. jejuni challenge. The prophylactic probiotic treatments were effective only when very low challenge levels of C. jejuni were used. Otherwise, probiotic treatments failed to reduce C. jejuni colonization. Regardless of treatment, as birds further aged C. jejuni numbers soon approached the levels observed in the control birds. We sought an alternative approach, as commercial broilers may be exposed to infectious levels of C. jejuni at any time during production. Two of our most promising antagonistic isolates, Lactobacillus salivarius NRRL B-30514 and Paenibacillus polymyxa NRRL B-30509, were further studied for effectiveness in reducing C. jejuni in chickens. When 250 mg of purified bacteriocins (produced by these organisms)/kg feed were fed therapeutically to chickens colonized with C. jejuni, colonization was reduced by at least one million-fold. Treatments with viable probiotic bacterial cultures were ineffective in reducing C. jejuni in chickens, while bacteriocin treatment from these corresponding bacteria substantially reduced C. jejuni colonization in the live birds.
Microbial Ecology in Health and Disease 07/2009; 20(2):74-79. DOI:10.1080/08910600802030196
[Show abstract][Hide abstract] ABSTRACT: Worldwide, dramatic numbers of reports document the increasing frequency of methicillin resistant Staphylococcus aureus (MRSA) infections. Other clinically significant human pathogens are increasingly recognized as unresponsive to therapeutic antibiotics of last resort. These previously treatable infections now account for unfortunate numbers of human disease and deaths following severe surface wounds or exposure to nosocomial sources. The inexorable and inextricable expansion of antibiotic resistant pathogens requires a new and viable response. This report documents a novel set of bacteriocins capable of inactivating MRSA and other refractile, antibiotic-resistant bacterial infections.
[Show abstract][Hide abstract] ABSTRACT: An effective bacteriocin was identified and characterized. Lactic acid bacteria were screened against Campylobacter jejuni. One bacteriocin producer, Enterococcus faecium (NRRL B-30746), was studied. The isolate was grown, and the bacteriocin was purified to single-band homogeneity. Biochemical traits indicated that the peptide was a Class IIa bacteriocin, and it was named E 50-52. The bacteriocin had a molecular weight of 3339.7 and an isoelectric point of 8.0. The minimal inhibitory concentrations of E 50-52 against C. jejuni, Yersinia spp., Salmonella spp., Escherichia coli O157:H7, Shigella dysenteriae, Morganella morganii, Staphylococcus spp., and Listeria spp. ranged from 0.025 to 32 microg/mL. In therapeutic broiler trials, oral treatment with E 50-52 reduced both C. jejuni and Salmonella enteritidis by more than 100,000-fold in the ceca, and systemic S. enteritidis was reduced in the liver and spleen. The wide range of antibacterial activity of bacteriocin E 50-52 against pathogens provides a promising alternative to antibiotics.
Journal of Agricultural and Food Chemistry 04/2008; 56(6):1942-8. DOI:10.1021/jf073284g · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Strain NRRL B-30745, isolated from chicken ceca and identified as Enterococcus durans, Enterococcus faecium, or Enterococcus hirae, was initially identified as antagonistic to Campylobacter jejuni. The isolate produced a 5,362-Da bacteriocin (enterocin) that inhibits the growth of Salmonella enterica serovar Enteritidis, S. enterica serovar Choleraesuis, S. enterica serovar Typhimurium, S. enterica serovar Gallinarum, Escherichia coli O157:H7, Yersinia enterocolitica, Citrobacter freundii, Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa, Proteus mirabilis, Morganella morganii, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Campylobacter jejuni, and 20 other Campylobacter species isolates. The enterocin, E-760, was isolated and purified by cation-exchange and hydrophobic-interaction chromatographies.
The proteinaceous nature of purified enterocin E-760 was demonstrated upon treatment with various proteolytic enzymes. Specifically,
the antimicrobial peptide was found to be sensitive to beta-chymotrypsin, proteinase K, and papain, while it was resistant
to lysozyme and lipase. The enterocin demonstrated thermostability by retaining activity after 5 min at 100°C and was stable
at pH values between 5.0 and 8.7. However, activity was lost below pH 3.0 and above pH 9.5. Administration of enterocin E-760-treated
feed significantly (P < 0.05) reduced the colonization of young broiler chicks experimentally challenged and colonized with two strains of C. jejuni by more than 8 log10 CFU. Enterocin E-760 also significantly (P < 0.05) reduced the colonization of naturally acquired Campylobacter species in market age broiler chickens when administered in treated feed 4 days prior to analysis.
[Show abstract][Hide abstract] ABSTRACT: Studies results about characteristics of growth medium for Legionella cultivation--legionelbacagar--are presented in article. It was shown that the medium has good growth characteristics and prolonged shelf life. Successful use of this medium for cultivation of Legionella pneumophila strains isolated during outbreak in Sverdlovsk region in 2007 was demonstrated. Feasibility of its use for isolation of Legionella from environment and clinical samples was discussed.
Zhurnal mikrobiologii, epidemiologii, i immunobiologii 01/2008;
[Show abstract][Hide abstract] ABSTRACT: Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin production.
[Show abstract][Hide abstract] ABSTRACT: Bacteriocins (Bcn) are natural peptides that are secreted by several taxonomically distant bacteria and exert bactericidal activity against other bacterial species. Their capacity to inhibit growth of virulent Mycobacterium tuberculosis H37Rv was evaluated in this study.
Five different Bcn were isolated and purified from bacterial culture supernatants, their amino acid sequence was determined, and activity against mycobacteria assessed in three different models: in vitro mycobacterial cultures, in vitro infection of mouse macrophages and in vivo high-dose infection of inbred mice.
In the in vitro model, four out of five Bcn exhibited stronger antimycobacterial activity than equal concentrations of a widely used anti-TB antibiotic, rifampicin. These Bcn were non-toxic for mouse macrophages at a concentration of 0.1 mg/L (>MIC(90) of these compounds). Pure Bcn did not inhibit mycobacterial growth within murine macrophages when added at 0.01-0.1 mg/L, suggesting that at physiologically tolerable concentrations these molecules do not penetrate through the membrane of eukaryotic cells. However, when administered as a complex with phosphatidylcholine-cardiolipin liposomes, Bcn5 (selected as a model compound due to its cytotoxicity and antimycobacterial activity regular titration curves) demonstrated capacity both to inhibit intracellular growth of M. tuberculosis and to prolong survival of mice in an acute TB model.
Given that the mechanism of Bcn bactericidal activity differs from that of all commonly used antibiotics, their possible involvement in complex TB therapies deserves further study.
[Show abstract][Hide abstract] ABSTRACT: We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was
termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes.
Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction
chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence
similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity
was stable following exposure to 90°C for 15 min, also consistent with these types of antibacterial peptides. The purified
protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of
nine isolation units; two groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit). At 7 days of age, one group of birds was treated with bacteriocin-emended feed for 3 days,
and one group was left untreated. At 10 days of age, the birds were sacrificed and the challenge strain was enumerated from
the bird cecal content. Bacteriocin treatment consistently reduced colonization at least one millionfold compared with levels
found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni. Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.
[Show abstract][Hide abstract] ABSTRACT: Campylobacter spp. cause numerous foodborne diseases. Poultry is thought to be a significant source of this zoonosis. Although many interventions designed to control this agent have been researched, none have succeeded. We evaluated a bacteriocin-based treatment to reduce Campylobacter jejuni colonization in poultry. A previously described purified bacteriocin (class IIa; molecular mass, 3,864 Da), secreted by Paenibacillus polymyxa NRRL-B-30509, was microencapsulated in polyvinylpyrrolidone, and 0.25 g of the purified bacteriocin was incorporated into 1 kg of chicken feed. One-day-old chickens were orally challenged and colonized with one of four isolates of C. jejuni, then reared in isolation facilities. Birds were provided ad libitum access to standard broiler starter feed and water for 7 days until 3 days before sampling, when only the treated groups of birds were provided the bacteriocin-emended feed described. In each of the eight (four by two replicates) trials, significant reductions in colonization by C. jejuni were observed (P < or = 0.05). As an example of this highly consistent data, in the first trial, 10 untreated 10-day-old chickens were colonized at a mean log 7.2 + 0.3 CFU/g of feces, whereas none of the 10 bacteriocin-treated 10-day-old chickens were colonized with detectable numbers of C. jejuni. Bacteriocin treatment dramatically reduced both intestinal levels and frequency of chicken colonization by C. jejuni. Feeding bacteriocins before poultry slaughter appears to provide control of C. jejuni to effectively reduce human exposure. This advance is directed toward on-farm control of pathogens, as opposed to the currently used chemical disinfection of contaminated carcasses.
Journal of food protection 08/2005; 68(7):1450-3. · 1.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We evaluated anti-Campylobacter activity among 365 Bacillus and Paenibacillus isolates from poultry production environments. One novel antagonistic Bacillus circulans and three Paenibacillus polymyxa strains were identified and further studied. Cell-free ammonium sulfate precipitate (crude antimicrobial preparation) was obtained from each candidate culture. Zones of Campylobacter growth inhibition surrounding 10 microl of this crude antimicrobial preparation were quantified using a spot test. Campylobacter growth resumed when the preparation was preincubated with selected protease enzymes, demonstrating peptide characteristics consistent with a bacteriocin. These peptides were further purified using combinations of molecular mass resolution and ion exchange chromatography. Molecular masses of the peptides were estimated at approximately 3,500 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing was used to determine the pI values of the peptides. Amino acid sequences of the bacteriocins and more precise molecular masses were obtained by matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF) analysis. The bacteriocin from P. polymyxa NRRL B-30507 had a pI of 4.8, that from P. polymyxa NRRL B-30509 had a pI of 7.2, that from P. polymyxa NRRL B-30508 had a pI of 4.8, and that from B. circulans NRRL B-30644 had a pI of 7.8. The amino acid sequences were consistent with those of class IIa bacteriocins. These antagonists and the corresponding bacteriocins may be useful in the control of Campylobacter infection in poultry.
Journal of food protection 02/2005; 68(1):11-7. · 1.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antibioticograms of enterohemorrhagic strains of serogroup O157 Escherichia coli isolated in the Russian Federation and Japan were comparatively studied. Strains with multiple drug resistance were detected. The main biochemical characteristics of the isolates were investigated. Significant differences in susceptibility spectra of the isolates and in their fermentative properties were revealed.
Antibiotiki i khimioterapii͡a = Antibiotics and chemoterapy [sic] / Ministerstvo meditsinskoĭ i mikrobiologicheskoĭ promyshlennosti SSSR 02/1998; 43(11):16-20.
[Show abstract][Hide abstract] ABSTRACT: A highly sensitive latex test system for identification of Legionella in the external medium and clinical materials have been designed. Protein antigens and polysaccharide components of the outer membrane of the agent were analyzed. Proteins having a molecular mass of 45, 29, and 24 kDa, as well as a polysaccharide component of LPS were found to be common for all L. pneumophila species. Highly affinic immunoglobulins to the antigenic components obtained were covalently linked with latex particles. The test system developed does not give cross-reactions with other microorganisms. The sensitivity of the system is 10(4) COE/ml. Testing water and clinical material samples confirmed that the developed system is more sensitive than the bacteriological method and the direct fluorescence test. In addition, the system is simple to use, cost-effective, it requires little time (no more than 5 min).
Vestnik Rossiĭskoĭ akademii meditsinskikh nauk / Rossiĭskaia akademiia meditsinskikh nauk 02/1997;