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.

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 log 10 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.Microorganisms produce a variety of compounds which demonstrate antibacterial properties. One group of these compounds, the bacteriocins, consists of relatively small bactericidal peptides. The widespread occurrence of bacteriocins in bacterial species isolated from complex microbial communities, such as the intestinal tract, oral surfaces, or other epithelial surfaces, suggests that they may have a regulatory role in terms of population dynamics within bacterial ecosystems. Bacteriocins are defined as compounds produced by bacteria that have a biologically active protein moiety and bactericidal action (51). In recent years, a renewed interest in bacteriocinlike activities has led to the discovery, isolation, and purification of bacteriocins from both gram-negative and gram-positive organisms (28). They are now being considered for a variety of antimicrobial uses in foods and medicine (17, 41).Lactic acid bacteria (LAB) are among the most well known and investigated producers of microbial antagonists. These include the well-characterized bacteriocins (6, 30, 55), potential bacteriocinlike substances (57), and other antagonists not necessarily related to bacteriocins (24,36,44). The LAB are gram-positive, non-spore-forming, catalase-negative organisms devoid of cytochromes. They are anaerobic...

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.

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.

The unacceptably high frequency of Campylobacter jejuni transmission from poultry to humans encourages scientists to consider and create alternative intervention strategies to control the pathogen in poultry production. Extremely high numbers of Campylobacter (often >10(8) cfu/g of poultry intestinal material) potentiate high numbers of the organism on the processed broiler carcass with increasing consequent human health risk. Many scientists believe interventions during poultry production portend the greatest opportunity for reducing risk of disease. Over the past 10 yr, we have focused our studies on nonantibiotic bacteriocin application to intervene during animal production and this is the subject of the current review. The application of therapeutic bacteriocin treatments to reduce poultry colonization diminishes Campylobacter from >10(8) cfu/g of cecal materials to nondetectable or very low levels in treated birds. Further, the review provides scientists with a useful starting point for the further development of industry-applicable interventions leading to reduced transmission of this agent in human disease.

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.

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 hydrophobicinteraction 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 g/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 log 10 and S. Enteritidis counts in both the liver and the spleen of treated birds were reduced by 6 to 8 log 10 /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.

BackgroundThe spread of carbapenemase-producing Enterobacteriaceae (CPE) is a great problem of healthcare worldwide. Study of the spread for blaOXA-48-like genes coding epidemically significant carbapenemases among hospital pathogens is important for the regional and global epidemiology of antimicrobial resistance.MethodsAntibacterial resistant isolates of Klebsiella pneumoniae (n = 95) from 54 patients, P.mirabilis (n = 32) from 20 patients, Enterobacter aerogenes (n = 6) from four patients, and Enterobacter cloacae (n = 4) from four patients were collected from January, 2013 to October, 2014 in neurosurgical intensive care unit (ICU) of the Burdenko Neurosurgery Institute, Moscow. Characteristics of the isolates were done using susceptibility tests, PCR detection of the resistance genes, genotyping, conjugation, DNA sequencing, and bioinformatic analysis.ResultsMajor strains under study were multi drug resistant (MDR), resistant to three or more functional classes of drugs simultaneously—98.9 % K. pneumoniae, 100 % P.mirabilis, one E.aerogenes isolate, and one E.cloacae isolate. Molecular-genetic mechanism of MDR in K.pneumoniae and P.mirabilis isolates were based on carrying of epidemic extended-spectrum beta-lactamase blaCTX-M-15 gene (87.2 and 90.6 % accordingly), carbapenemase blaOXA-48-like gene (55.3 and 23.3 % accordingly), and class 1 (54.8 and 31.3 % accordingly) and class 2 (90.6 % P. mirabilis) integrons. The blaOXA-48-like-positive K. pneumoniae were collected during whole two-year surveillance period, while P. mirabilis and Enterobacter spp. carrying blaOXA-48-like genes were detected only after four and 18 months after the research start, respectively. The blaOXA-48-like gene acquisition was shown for P. mirabilis isolates collected from five patients and for E. cloacae isolate collected from one patient during their stay in the ICU, presumably from blaOXA-48-like-positive K. pneumoniae. The source of the blaOXA-244 gene acquired by E. aerogenes isolates and the time of this event were not recognized.ConclusionsThe expanding of CPE in the surveyed ICU was associated with the spread of blaOXA-48 and blaOXA-244 carbapenemase genes documented not only among K.pneumoniae, well-known bacterial host for such genes, but among P.mirabilis, E.aerogenes, and E. cloacae.