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 × 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

Identification of production challenges and benefits using value chain mapping of egg food systems in Nairobi, Kenya

Commercial layer and indigenous chicken farming in Nairobi and associated activities in the egg value chains are a source of livelihood for urban families. A value chain mapping framework was used to describe types of inputs and outputs from chicken farms, challenges faced by producers and their disease control strategies. Commercial layer farms were defined as farms keeping exotic breeds of chicken, whereas indigenous chicken farms kept different cross breeds of indigenous chicken. Four focus group discussions were held with producers of these chickens in peri-urban area: Dagoretti, and one informal settlement: Kibera. Qualitative data were collected on interactions between farmers, sources of farm inputs and buyers of poultry products, simple ranking of production challenges, farmers' perception on diseases affecting chicken and strategies for management of sick chicken and waste products. Value chain profiles were drawn showing sources of inputs and channels for distribution of chicken products. Production challenges and chicken disease management strategies were presented as qualitative summaries. Commercial layer farms in Dagoretti kept an average of 250 chickens (range 50–500); while flock sizes in Kibera were 12 chickens (range 5–20). Farms keeping indigenous chicken had an average of 23 chickens (range 8–40) in Dagoretti, and 10 chickens (range 5–16) in Kibera. Commercial layer farms in Dagoretti obtained chicks from distributors of commercial hatcheries, but farms in Kibera obtained chicks from hawkers who in turn sourced them from distributors of commercial hatcheries. Indigenous chicken farms from Dagoretti relied on natural hatching of fertilised eggs, but indigenous chicken farms in Kibera obtained chicks from their social connection with communities living in rural areas. Outlets for eggs from commercial layer farms included local shops, brokers, restaurants and hawkers, while eggs from indigenous chicken farms were sold to neighbours and restaurants. Sieved chicken manure from Dagoretti area was fed to dairy cattle; whereas non-sieved manure was used as fertilizer on crops. Production challenges included poor feed quality, lack of space for expansion, insecurity, occurrence of diseases and lack of sources of information on chicken management. In Kibera, sick and dead chickens were slaughtered and consumed by households; this practice was not reported in Dagoretti. The chicken layer systems contribute to food security of urban households, yet they have vulnerabilities and deficiencies with regard to disease management and food safety that need to be addressed with support on research and extension

A search for new molecular targets for optimizing plague preventive vaccination and therapy

The causative agent of plague, Yersinia pestis, is a highly virulent bacterial pathogen and a potential bioweapon. Depending on the route of infection, two prevalent forms of the disease — bubonic and pneumonic, are known. The latter is featured by a high fatality rate. Mortality in untreated bubonic plague patients reaches up to 40—60%, whereas untreated pneumonic plague is always lethal. The development of the infectious process in susceptible host is accounted for by a whole set of pathogenicity factors in plague pathogen displaying various functional modalities being expressed depending on stage of infectious process, providing their coordinated expression. Knocking out any of such factors, in turn, may not either affect microbe virulence or lead to its attenuation. A search for new Yersinia pestis pathogenicity factors and subsequent development of highly effective subunit and live attenuated plague vaccines inducing development of pronounced cellular and humoral immune reactions, and/or assessment of their potential use as molecular targets for plague therapy still remain a pressing issue, as both currently licensed plague vaccines do not meet the WHO requirements, whereas strains of plague microbe isolated in Madagascar are resistant to all drugs recommended for plague antibacterial therapy. Here we summarize an impact of described and newly discovered pathogenicity factors into the virulence of Y. pestis strains and their protective anti-plague activity. An effect of loss of genes encoding regulatory proteins as well as mutations in the genes for various transport systems of Y. pestis on attenuation of virulent strains is described as well. Perspectives for introducing characterized antigens into prototype subunit vaccine as well as some other obtained mutants into prototypes of living attenuating vaccines were assessed. The use of antibiotics for plague treatment has been embraced by the World Health Organization Expert Committee on Plague as the “gold standard” treatment. However, concerns regarding development of antibioticresistant Y. pestis strains accounted for further exploring alternatives to plague therapy. Several research groups continue to seek for other alternative approaches, e. g. treatment with inhibitors of pathogenicity factors. Preliminary data attempting to treat plague patients with pathogenicity factor inhibitors are summarized. Antivirulence drugs targeting key microbial factors represent new promising therapeutic options in the fight against antibiotic-resistant bacteria

Functional characterization and biological significance of Yersinia pestis lipopolysaccharide biosynthesis genes

The Gram negative bacterium Yersinia pestis is the etiological agent of flea transmitted fulminant systemic rodent zoonosis and the reason of the three devastating pandemics of plague Lipopolysaccharide (LPS, endotoxin) is an impor tant factor of pathogenicity of Gram negative bacteria. The full LPS molecule (S form LPS) consists of three well defined domains: i) lipid A composed of sugars, fatty acids, and phosphate; it represents the endotoxic princi ple of the LPS and anchors it in the outer membrane; ii) a core oligosaccharide containing charged groups; and iii) an O specific polysaccharide (O antigen), which carries ISSN 0006 2979, Biochemistry (Moscow), 2011, Vol. 76, No. 7, pp. 808 822. © Pleiades Publishing, Ltd., 2011. Published in Russian in Biokhimiya, 2011, Vol. 76, No. 7, pp. 989 1005 Abstract-In silico analysis of available bacterial genomes revealed the phylogenetic proximity levels of enzymes responsible for biosynthesis of lipopolysaccharide (LPS) of Yersinia pestis, the cause of plague, to homologous proteins of closely relat ed Yersinia spp. and some other bacteria (Serratia proteamaculans, Erwinia carotovora, Burkholderia dolosa, Photorhabdus luminescens and others). Isogenic Y. pestis mutants with single or double mutations in 14 genes of LPS biosynthetic path ways were constructed by site directed mutagenesis on the base of the virulent strain 231 and its attenuated derivative. Using high resolution electrospray ionization mass spectrometry, the full LPS structures were elucidated in each mutant, and the sequence of monosaccharide transfers in the assembly of the LPS core was inferred. Truncation of the core decreased sig nificantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less effi cient incorporation of 4 amino 4 deoxyarabinose into lipid A. Impairing of LPS biosynthesis resulted also in reduction of LPS dependent enzymatic activities of plasminogen activator and elevation of LD 50 and average survival time in mice and guinea pigs infected with experimental plague. Unraveling correlations between biological properties of bacteria and partic ular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague

Dynamics of Antibody Response to <i>Yersinia pestis</i> Proteins in Plague Affected Guinea Pigs

Designing of new means for the specific prevention of plague, especially protein subunit vaccines, is impossible without studying the role of individual antigens in the manifestation of the pathogenic and immunogenic properties of Yersinia pestis. The aim of the present study was to determine the antibody levels to Y. pestis antigens in guinea pigs that survived infection with sub-lethal doses of virulent plague agent strains using enzyme immunoassay (ELISA). Materials and methods. Guinea pigs were inoculated subcutaneously with 30 CFU of the wild type Y. pestis subsp. Pestis strain 231 or non-capsular Y. pestis subsp. pestis Caf1-negative strain 358/12. Blood samples from sick or recovered guinea pigs were collected on day 15, 30, 60, and 90 after infection. The antibody response was assessed by 18 recombinant Y. pestis proteins in ELISA. Results and discussion. Heterogeneity of the antibody responses to the majority of the antigens with variation of IgG titers from animal to animal has been revealed. We observed increase in antibody titers by day 90 for the most analyzed antigens in the sera of the guinea pigs injected with wild type Y. pestis 231. On the contrary we found reduction in antibody titers by day 90 in case of inoculation with Y. pestis 358/12. The preservation of antibodies to Y. pestis proteins of different localization in the organism of the guinea pigs, as well functional activity, and the degree of representation on the surface of bacterial cell for a prolonged period of time indicates the multiplex nature of the plague immunity formation. Our findings are significant for the future design and development of effective vaccines against plague and the search for new targets for diagnostics of this disease

TYPLEX® Chelate, a novel feed additive, inhibits Campylobacter jejuni biofilm formation and cecal colonization in broiler chickens

Reducing Campylobacter spp. carriage in poultry is challenging, but essential to control this major cause of human bacterial gastroenteritis worldwide. Although much is known about the mechanisms and route of Campylobacter spp. colonization in poultry the literature is scarce on antibiotic-free solutions to combat Campylobacter spp. colonization in poultry. In vitro and in vivo studies were conducted to investigate the role of TYPLEX® Chelate, a novel feed additive, in inhibiting Campylobacter jejuni (C. jejuni) biofilm formation and reducing C. jejuni and Escherichia coli (E. coli) colonization in broiler chickens at market age. In an in vitro study, the inhibitory effect on C. jejuni biofilm formation using a plastic bead assay was investigated. The results demonstrated that TYPLEX® Chelate significantly reduces biofilm formation. For in vivo study, 800 broilers (one-day old) were randomly allocated to 4 dietary treatments in a randomised block design, each having 10 replicate pens with 20 birds per pen. At day 21, all birds were challenged with C. jejuni via seeded litter. At day 42, caecal samples were collected and tested for volatile fatty acid (VFA) concentrations, C. jejuni and E. coli counts. The results showed that TYPLEX® Chelate reduced the carriage of C. jejuni and E. coli in poultry by 2 and 1 log₁₀ per gram caecal sample, respectively, and increased caecal VFA concentrations. These findings support TYPLEX® Chelate as a novel non-antibiotic feed additive that may help produce poultry with a lower public health risk of Campylobacteriosis

S-layer protein 2 of 'Lactobacillus crispatus' 2029, its structural and immunomodulatory characteristics and roles in protective potential of the whole bacteria against foodborne pathogens

We have previously demonstrated that human vaginal Lactobacillus crispatus 2029 (LC2029) strain is highly adhesive to cervicovaginal epithelial cells, exhibits antagonistic activity against genitourinary pathogens and expresses surface-layer protein (Slp). The aims of the present study were elucidation of Slp structural and immunomodulatory characteristics and its roles in protective properties of the whole vaginal LC2029 bacteria against foodborne pathogens. Enteric Caco-2 and colon HT-29 cell lines were used as the in vitro models of the human intestinal epithelial layer. LC2029 strain has two homologous surface-layer (S-layer) genes, slp1 and slp2. Whilst we found no evidence for the expression of slp1 under the growth conditions used, a very high level of expression of the slp2 gene was detected. C-terminal part of the amino sequence of Slp2 protein was found to be highly similar to that of the conserved C-terminal region of SlpA protein of L. crispatus Zj001 isolated from pig intestines and CbsA protein of L. crispatus JCM5810 isolated from chicken intestines, and was substantially variable at the N-terminal and middle regions. The amino acid sequence identity between SlpA and CbsA was as high as 84%, whilst the identity levels of these sequences with that of Slp2 were only 49% and 50% (respectively). LC2029 strain was found to be both acid and bile tolerant. Survival in simulated gastric and intestinal juices of LC2029 cells unable to produce Slp2 was reduced by 2-3 logs. Vaginal L. crispatus 1385 (LC1385) strain not expressing Slp was also very sensitive to gastric and intestinal stresses. Slp2 was found to be non-covalently bound to the surface of the bacterium, acting as an adhesin and facilitating interaction of LC2029 lactobacilli with the host immature or fully differentiated Caco-2 cells, as well as HT-29 cells. No toxicity to or damage of Caco-2 or HT-29 epithelial cells were detected after 24 h of colonization by LC2029 lactobacilli. Both Slp2 protein and LC2029 cells induced NF-kB activation in Caco-2 and HT-29 cells, but did not induce expression of innate immunity mediators Il-8, Il-1β, and TNF-α. Slp2 and LC2029 inhibited Il-8 production in Caco-2 and HT-29 cells induced by MALP-2 and increased production of anti-inflammatory cytokine Il-6. Slp2 inhibited production of CXCL1 and RANTES by Caco-2 cells during differentiation and maturation process within 15 days. Culturing Caco-2 and HT-29 cells in the presence of Slp2 increased adhesion of bifidobacteria BLI-2780 to these enterocytes. Upon binding to Caco-2 and HT-29 cells, Slp2 protein and LC2029 lactobacilli were recognized by toll-like receptors (TLR) 2/6. It was shown that LC2029 strain is a strong co-aggregator of foodborne pathogens Campylobacter jejuni, Salmonella enteritidis, and Escherichia coli O157:H used in this study. The Slp2 was responsible for the ability of LC2029 to co-aggregate these enteropathogens. Slp2 and intact LC2029 lactobacilli inhibited foodborne pathogen-induced activation of caspase-9 and caspase-3 as apoptotic biomarkers in Caco-2 and HT-29 cells. In addition, Slp2 and Slp2-positive LC2029 strain reduced adhesion of tested pathogenic bacteria to Caco-2 and HT-29 cells. Slp2-positive LC2029 strain but not Slp2 alone provided bactericidal effect on foodborne pathogens. These results suggest a range of mechanisms involved in inhibition of growth, viability, and cell adhesion properties of pathogenic Proteobacteria by the Slp2 producing LC2029, which may be useful in treatment of necrotizing enterocolitis (NEC) in newborns and foodborne infectious diseases in children and adults, increasing the colonization resistance and maintaining the intestinal homeostasis

Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae