A study of antibiotic and disinfectant susceptibility of Elizabethkingia meningoseptica.

For the local health service, Elizabethkingia meningoseptica remains a relatively new and little-known pathogen, whereas in many countries of Europe, Asia and other continents it is considered as a potential causative agent of nosocomial infections, especially in premature infants and immunocompromised patients. An analysis of the literature data, as well as our results indicate that E. meningoseptica should be considered as a potential pathogen, which is characterized by a unique profile of susceptibility to antimicrobial agents (AMP) and disinfectants. This article presents the results of a study of susceptibility to AMP and disinfectants of three isolates of E. meningoseptica, isolated during an investigation of an outbreak in one of the perinatal centers of the Russian Federation, where three cases of sepsis with a fatal outcome in premature infants caused by co-infection with Acinetobacter baumannii and E. meningoseptica were recorded between January and February 2016.

[Phenotypic and molecular genetic properties of Escherichia coli clinical strains isolated from patients with urological diseases].

OBJECTIVE: Microbiological and molecular genetic characterization resistance profiles of Escherichia coli strains isolated in a pilot single-center clinical study from patients of the urological department in Yaroslavl in 2016-2017. MATERIALS AND METHODS: Clinical strains of E. coli (n=18) were isolated from the urine of women aged 23-84 years. The mobility of bacteria, colicinogenicity, and sensitivity to lactobacilli antagonism, biofilm formation, and susceptibility to antimicrobials were evaluated. The antibiotic resistance genes were identified. RESULTS: The E. coli strains had a wide heterogeneity in mobility, colicinogenicity, and biofilm formation. They were sensitive to Lactobacillus acidophilus antagonism, as well as to nitrofurantoin, meropenem, fosfomycin and the main functional classes of disinfectants and antiseptics, but are resistant to beta-lactams, fluoroquinolones and aminoglycosides. The mcr-1 gene providing resistance to colistin was identified in two strains. CONCLUSIONS: Analysis of genetic antibiotic resistance determinants revealed the genetic diversity of clinical E. coli strains. The obtained data on the strain sensitivity to antibacterials and disinfectants can be used by clinicians in choosing the optimal antibiotic therapy and treatment of abiotic surfaces in urological departments.

[Diagnostic bacteriophage V32 as a tool for the rapid identification of Escherichia coli serogroup O157.]

Bacteriophage V32, a representative of bacterial viruses of the Myoviridae family Ounavirinae subfamily, is proposed for search and identification of E. coli O157 serogroup, including Shiga-toxin producing E. coli O157:H7 (STEC O157:H7), among cultures of enterobacteria from the primary seeding of the material studied. Phage genome containes a linear double-stranded DNA of 87875 base pairs with G/C-content of 38.9% and includes 132 open reading frames (ORF). In the genome, there are no determinants of antibiotic resistance, virulence genes of STEC and other well-known pathogroups of E. coli. It has been established that phage V32 has lytic activity against all studied cultures of E. coli O157 serogroup (n=183) isolated from people and farm animals in various regions of the Russian Federation, as well as in Japan and Italy. At the same time, the phage lyses only 6 of 182 strains (3.3%) of E. coli not belonging to the O157 serogroup and is not active against strains of other enterobacteria. That is, the phage has a high specificity. The use of bacteriophage V32 as a diagnostic tool is a highly efficient, fast, cheap and simple method for identifying E. coli serogroup O157, including the serotype E. coli O157: H7, in any bacteriological laboratory without special equipment and special training of performers.

[The characteristic of diarrheagenic Escherichia separated from children aged under 5 years old in Yaroslavl.]

The diarrheagenic bacteria coli take a significant place among agents of acute intestinal infections in children aged under 5 years. The main danger among these pathogens is represented by both enterotoxigenic E. coli causing enteritis and enterocolitis accompanied by acute dehydration diarrhea and Escherichia producing shiga-toxin being agents of hemorrhagic colitis and hemolytic uremic syndrome. The fast and proper identification of agents of these two groups of pathogens is an important task of bacteriologists to be resolved for successful treatment of patient because tactics of therapy of enterotoxigenic diarrhea and hemorrhagic colitis and hemolytic uremic syndrome significantly differ. The high capacity of Escherichia coli to form populations resistant to anti-microbial medications, including pan-resistant ones, also is a serious problem for science and public health. The object of study was a collection of isolates of E. coli (n = 112), separated from 112 children aged under 5 years with clinical manifestations of acute intestinal infection, food toxic infection hemocolitis and diarrhea of obscure etiology in Yaroslavl in 2015-2017. Initially, the strains of E. coli were tested using diagnostic agglutinating coli-serums and then using reagents' kit «AmpliSens®Escherichiosis-FL¼ for detection and differentiation DNAof diarrheagenic bacteria coli and also with specific oligonucleotide primers to genes of virulence and O-serum group belonging. The obtained data permitted to determine belonging of analyzed strains of E. coli to four sub-groups: ЕНЕС (n = 9), EPEC (n = 17), ETEC (n = 1) и EAgEС (n = 1). All of them were agents of genes of pathogenicity specific for every pathogroup. The most numerous group EPEC was represented by strains of five serogroups with dominating among them serogroup O26 (9 strains). Therefore, studying collection of strains of diarrheagenic Escherichia isolated during 2015-1017 in Yaroslavl from children aged under 5 years with acute intestinal infections permitted to demonstrate efficiency of application of molecular genetic methods of analysis for characterizing E. coli i.e. establishment their serogroups, detection of genes of virulence and attributing to pathogroups.

MOLECULAR TYPING OF SHIGELLA STRAINS.

Shigelloses still provide one of the toughest health challenges in many countries, especially in developing ones. Current molecular-genetic techniques are increasingly replacing conventional phenotypic approaches to investigating Shigella-associated infections. The review describes available methods of typing Shigella bacteria, their potential applications and selection criteria depending on the research task. It also deals with their advantages and disadvantages. Such methods as PFGE (pulsed field gel electrophoresis), the "gold standard" in typing various pathogens, and MLVA (multi-locus variable number of tandem repeat analysis) are the most widely used Shigella typing procedures. These methods are often combined or used along with some other genotyping tools. In the nearest future, with the advent of web platforms allowing express processing data, whole genome sequence will possibly become more widely used in lab diagnostics to evaluate outbreaks and to perform epidemiological monitoring.

Phagebiotics in treatment and prophylaxis of healthcare-associated infections.

We have developed a phagebiotic composition using 8 virulent bacteriophages (2 strains of each species) which are able to lyse Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. The unique character of the developed composition is ensured by particular properties of each bacteriophage comprising the preparation, including their range of lytic activity toward specific bacterial pathogens, morphology of their plaques, cycle of their development, restriction profile of their DNAs, specificity of their genomes (based on complete genome sequencing), and other properties. The preparation did not produce any signs of acute or chronic intoxication in the experimental animals. Therapeutic and prophylactic efficiency of the phagebiotic composition was demonstrated in the prevention and treatment of the experimental acute K. pneumoniae infection in mice. The investigations have shown that the preparation possesses a high therapeutic efficiency and is highly competitive with ciprofloxacin which is very effective against the infective strain K. pneumoniae. Our small-scale clinical trial was aimed to evaluate therapeutic effectiveness of the phagebiotic composition in an epidemiological emergency situation in an intensive care unit, caused by multi-resistant strains of Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. Seventy nine per cent of the initial samples from 14 patients' endotracheal aspirate, blood and urine were contaminated. Twenty-four hours after the 3-day phage therapy (20 ml of cocktail at a titer for each phage 108 pfu/ml were introduced intragastrically through a tube once a day) contamination level dropped to 21%. Hence the obtained results enabled us to create a new phagebiotic composition that may be used as an alternative to antibiotics to treat these healthcare-associated infections.

A small-scale experiment of using phage-based probiotic dietary supplement for prevention of E. coli traveler's diarrhea.

Traveler's diarrhea (TD) is caused by Escherichia coli in 30% of cases. We have developed a phage cocktail for prophylaxis of TD caused by E.coli, Shigella flexneri, Shigella sonnei, Salmonella enterica, Listeria monocytogenes or Staphylococcus aureus, and investigated its effectiveness against infection caused by the non-pathogenic Lac (-) strain of E.coli K12 C600 in animal and human trials. On the 6th day of both animal and human trials E. coli K12 C600 strain was detected in titer of 104 CFU/g of mice feces and 106 CFU/g of human feces in the control (untreated) groups, while it was not detected in the samples of either of the study (phage-treated) groups. These results have great significance because the original coliphages included in the cocktail have a broad host-range including ETEC, EAEC and EHEC strains which cause severe cases of TD.

[Molecular-genetic characterization of shiga-toxin producing Escherichia coli isolated during a food-borne outbreak in St. Petersburg in 2013].

UNLABELLED: Shiga toxin-producing Escherichia coli (STEC) food-borne infections are reported worldwide and represent a serious problem for public healthcare. In the Russian Federation there is little information on epidemiology and etiology of STEC-infections as well as on molecular-genetic peculiarities of STEC pathogens. OBJECTIVE: Our aim was to describe a food-borne outbreak as hemorrhagic colitis (HC) along with hemolytic uremic syndrome (HUS), enterocolitis, and acute gastroenteritis in children in St. Petersburg in 2013. METHODS: Epidemiological, microbiological, molecular-genetic and bioinformatic methods were applied. RESULTS: Objects to study were clinical specimens, milk and food samples, as well as STEC strains isolated during the outbreak. The outbreak of food-borne infection was found to be caused by STEC-contaminated raw milk as confirmed by epidemiological analysis, detection of STEC DNA and isolation of relevant pathogens in milk and sick children fecal specimens. The whole-genome sequencing revealed two groups ofpathogens, E. coli O157:H7 and E. coli O101:H33 among collected strains. Group I strains were attributed to the previously known sequence type ST24, while group II strains belonged to the previously non-described sequence type ST145. In strain genomes of both groups there were identified nucleotide sequences of VT2-like prophage carrying stx2c gene, plasmid enterohemolysin gene, and gene of the STEC main adhesion factor intimin. Gene of intimin gamma was identified in E. coli O157:H7 strains and intimin iota 2 in E. coli O101:H33 strains. The latter previously was identified only in enteropathogenic E. coli (EPEC) strains. CONCLUSION: The additional knowledge of epidemiology and biology of STEC pathogens would assist clinicians and epidemiologists in diagnosing, treating and preventing hemorrhagic colitis.

[Antimicrobial activity of bacteriocin S760 produced by Enterococcus faecium strain LWP760].

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.

[Efficacy of enterocin S760 in treatment of mice with anthrax infection due to Bacillus anthracis M-71].

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.

[An algorithm for identification of CTX-M-type beta-lactamase genes using restriction fragment length polymorphism analysis of PCR-product].

The algorithm of the identification of the bla(CTX-M) genes coding CTX-M-type beta-lactamases providing resistance to cephalosporins III-IV was developed. This algorithm provides identification of 49 genes of 96 genes presented in the GenBank database so far. Remaining 47 genes can be identified as consisting of small sub-groups composed of 2-6 genes with the exception of sub-group of the bla(CTX-M-14)-like genes composed of 13 genes. The identification of the bla(CTX-M) genes is based on two-step restriction fragment length polymorphism analysis of 544 bp PCR-product (PCR-RFLP). In the first step, determination of subtype (cluster) of the bla(CTX-M) gene occurred using the restriction nuclease Alu I: cluster 1, -2, -8, -9 or -25. Moreover, four genes can be identified just at this step: bla(CTX-M)-59, (cluster 2); bla(CTX-M-63) (cluster 8), bla(CTX-M-45) (cluster 9), and bla(CTX-M-78) (hybrid gene between cluster 2 and cluster 25). At the second step gene identification goes on inside of each cluster separately using a set of 26 restriction nucleases. As a result of the PCR-RFLP-analysis, 23 bla(CTX-M) genes can be identified at the cluster 1, 11 genes--at the cluster 2, 4 genes--at the cluster 8, 9 genes--at the cluster 9, 1 gene--at the cluster 25, and 2 hybrid genes: bla(CTX-M-78) (between clusters 2 and 25), and bla(CTX-M-64) (between clusters 1 and 9). The described algorithm was used for identification of the blac(CTX-M) genes (n = 585) detected in Enterobacteriaceae nosocomial isolates (n = 877), collected from Russial hospitals in 2003-2007. It was shown that major genes belonged to cluster 1 (n = 543), namely--bla(CTX-M-15) gene (n = 515), bla(CTX-M-3) (n = 25), bla(CTX-M-22) (n = 1), bla(CTX-M-23) (n = 1), and bla(CTM-34) (n = 1). Moreover, the genes atributed to cluster 2 were identified: bla(CTX-M-2) (n = 1), and bla(CTX-M-5) (n = 4); and genes belonged to cluster 9: bla(CTX-M-9) (n = 2), and bla(CTX-M-14) (n = 35).

[Use of enterocin S760 for prevention and treatment of experimental Salmonella infection in mice].

AIM: To demonstrate treatment efficacy of bacteriocin S760 synthesized by Enterococcus faecium 760 for septic Salmonella infection in mice. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSION: Obtained results demonstrated high treatment efficacy of bacteriocin S760 during septic salmonellosis and perspectives of its use in medicine and animal health.

Bacteriocins to control Campylobacter spp. in poultry--A review.

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.

[Genetic determinants of antibacterial resistance among nosocomial Escherichia coli, Klebsiella spp., and Enterobacter spp. isolates collected in Russia within 2003-2007].

Nosocomial bacterial isolates collected within 2003-2004 (n=411) and 2005-2007 (n=422) were highly resistant to cephalosporins III-IV and antibacterials of other groups (aminoglycosides, fluoroquinolons, chloramphenicol, and co-trimoxazole). Genes encoding TEM, SHV, CTX-M, OXA-2, and AmpC types of beta-lactamases (BLs) in the E. coli, Klebsiella spp., and Enterobacter spp. isolates were detected using polymerase chain reaction (PCR). Prevalent CTX-M-type BLs were detected in 85% of the E. coli, 87% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the first strain collection and in 94% of the E. coli, 91% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the second one. Genes belonging to three subtypes of blacTx-M genes were identified: bla(CTX-M-1) (228 bla(CTX-M-15) and six bla(CTX-M-3) of the first strain collection; 275 bla(CTX-M-15), three bla(CTX-M-3), and one bla(CTX-M-22) of the second one), bla(CTX-M-2) (one bla(CTX-M-5) of the first strain collection and one bla(CTX-M-2) of the second one), bla(CTX-M-9) (17 bla(CTX-M-14) and one bla(CTX-M-9) of the first strain collection; seven bla(CTX-M-14) and one bla(CTX-M-9) of the second one). Three isolates of the first strain collection and one isolate of the second one carried two genes belonging to two different subtypes, i.e., bla(CTX-M-15) and bla(CTX-M-14) simultaneously. The bacterial isolates had high levels of associative resistance to ciprofloxacin, co-trimoxazole, gentamicin, amikacin, and chloramphenicol associated with the resistance gene cassettes aadA1, aadA2, aadA5, aadB, aacA4, aac(6')Ib; dfrA1, dfrA5, dfrA12, dfrA17, cmlA1, ereA2, and catB8 in the class 1 integrons and the resistance gene cassettes dfrA1, sat1, and aadA1 in the class 2 integrons.

[Genetic environments of bla(CTX-M) genes located on conjugative plasmids of Enterobacteriaceae nosocomial isolates collected in Russia within 2003-2007].

The study showed that bla(CTX-M) genes were present in the genomes of 71% of cephalosporin resistant Enterobacteriaceae nosocomial isolates (n=833) collected in Russian hospitals within 2003-2007, including 91% of E.coli, 90% of Klebsiella spp., 38% of Enterobacter spp., 31% of Citrobacter spp. (n=9), and 36% of the other Enterobacteriaceae species. The genes belonging to the following subtypes (clusters) were identified: bla(CTX-M-1) (529 bla(CTX-M-15) genes; 25 bla(CTX-M-3) genes; 1 bla(CTX-M-22) gene, 1 bla(CTX-M-23) gene, and 1 bla(CTX-M-34) gene); bla(CTX-M-2) (1 bla(CTX-M-2) gene, and 4 bla(CTX-M-5) genes), and bla(CTX-M-9) (2 bla(CTX-M-9) genes, and 28 bla(CTX-M-14) genes). It was shown that bla(CTX-M) genes were located on high-molecular weight (60-160 bp) conjugative plasmids belonging mainly to the incompatibility groups IncF, IncL/M and IncA/C (bla(CTX-M-15) gene); IncL/M(bla(CTX-M-3) gene); and IncF, IncL/Mand IncI1-ly (CTX-M-14 gene). The gene environments of bla(CTX-M) genes were shown specific for the subtype of the genes. A mobile genetic element ISEcp1 (in some cases deleted or inserted by IS26, IS1, IS10, resTn2, or resTn3 sequences, in direct or reverse position) were detected upstream of bla(CTX-M-3), bla(CTX-M-14), and bla(CTX-M-15) genes. A special characteristic was the sequence between ISEcp1 and bla(CTX-M) gene: 48 bp for bla(CTX-M-15) (except 1 E.coli isolate having such a sequence deleted by 3 bp); 127 bp for bla(CTX-M-3); 42 bp for bla(CTX-M-14). Downstream of bla(CTX-M) and bla(CTX-M-15) genes in the major bacterial isolates orf477 mucA and Delta orf477-Delta mucA sequences were detected respectively. Two isolates had additional Delta orf3 insertion inside of Delta orf477-Delta mucA sequence. Insertion sequence IS903 (intact or deleted) was detected downstream of bla(CTX-M-14) gene. Unlike the others, bla(CTX-M-2) and bla(CTX-M-9) genes were located inside of ISCR1 mobile element, downstream of class 1 integron and orf513 sequence.

Antimicrobial Activities of Bacteriocins E 50-52 and B 602 Against Antibiotic-Resistant Strains Involved in Nosocomial Infections.

The antimicrobial spectra of previously published bacteriocin E 50-52 (39 a.a.; 3,932 Da; pI = 8.5) and bacteriocin B 602 (29 a.a.; 3,864 Da; 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 B 602 ranged from ≤0.025-1.56 µg/ml, and for bacteriocin E 50-52 from 0.05 to 6.25 µg/ml for all of 64 bacterial clinical isolates tested. Interestingly, the bacteriocins studied demonstrate activity on both Gram-positive and Gram-negative bacteria. Bacteriocins E 50-52 and B 602 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.

Isolation and purification of enterocin E-760 with broad antimicrobial activity against gram-positive and gram-negative bacteria.

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 degrees 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.

Bacteriocins reduce Campylobacter colonization and alter gut morphology in turkey poults.

Campylobacter is a leading cause of food-borne illness in the United States. Recent evidence has demonstrated that bacteriocins produced by Bacillus circulans and Paenibacillus polymyxa reduce cecal Campylobacter colonization in broiler chickens infected with Campylobacter jejuni. As Campylobacter coli is the most prevalent Campylobacter isolate recovered in turkeys, the objectives of the present study were to evaluate the efficacy of these bacteriocins against C. coli colonization and their influence on the gastrointestinal architecture of young turkeys. In 3 separate trials, a total of 135 day-of-hatch poults (n = 45/trial) were orally challenged on d 3 with approximately 10(6) cfu of a mixture of 3 C. coli isolates. Immediately before bacteriocin treatment (d 10), cecal Campylobacter concentrations averaged 1.1 x 10(7) cfu/ g of cecal contents (n = 15/trial). On d 10 to 12 posthatch, 2 bacteriocin treatment groups were given free access to feed supplemented with purified, microencapsulated bacteriocins, whereas the positive control treatment group had access to untreated feed (n = 10/treatment group per trial). At the end of the 3-d dosing period, ceca and duodenal loops were collected for analysis. In each of the 3 separate trials, treatment with bacteriocin eliminated detectable ceca Campylobacter concentrations (detection limit, 1 x 10(2) cfu/g of cecal contents) vs. controls (1.0 x 106 cfu of Campylobacter/g of cecal contents). Duodenum crypt depth and goblet cell numbers were also reduced in turkeys treated with either bacteriocin vs. controls (P < 0.05). The dynamic reduction in crypt depth and goblet cell density in turkeys dosed with bacteriocin may provide clues to how bacteriocins inhibit enteric Campylobacter.

Isolation of a Lactobacillus salivarius strain and purification of its bacteriocin, which is inhibitory to Campylobacter jejuni in the chicken gastrointestinal system.

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 degrees 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.

The ability of flagellum-specific Proteus vulgaris bacteriophage PV22 to interact with Campylobacter jejuni flagella in culture.

BACKGROUND: There has been a recent resurgent interest in bacteriophage biology. Research was initiated to examine Campylobacter jejuni-specific bacteriophage in the Russian Federation to develop alternative control measures for this pathogen. RESULTS: A C. jejuni flagellum-specific phage PV22 from Proteus vulgaris was identified in sewage drainage. This phage interacted with C. jejuni by attachment to flagella followed by translocation of the phage to the polar region of the bacterium up to the point of DNA injection. Electron microscopic examination revealed adsorption of PV22 on C. jejuni flagella after a five minute incubation of the phage and bacteria. A different phenomenon was observed after incubating the mix under the same conditions, but for twenty minutes or longer. Phage accumulated primarily on the surface of cells at sites where flagella originated. Interestingly, PV22 did not inject DNA into C. jejuni and PV22 did not produce lytic plaques on medium containing C. jejuni cells. The constant of velocity for PV22 adsorption on cells was 7 x 10(-9) ml/min. CONCLUSION: It was demonstrated that a bacteriophage that productively infects P. vulgaris was able to bind C. jejuni and by a spot test that the growth of C. jejuni was reduced relative to control bacteria in the region of phage application. There may be two interesting applications of this effect. First, it may be possible to test phage PV22 as an antimicrobial agent to decrease C. jejuni colonization of the chicken intestine. Second, the phage could potentially be utilized for investigating biogenesis of C. jejuni flagella.