The crystal structure of a complex of Campylobacter jejuni dUTPase with substrate analogue sheds light on the mechanism and suggests the "basic module" for dimeric d(C/U)TPases

The crystal structure of the dUTPase from the important gastric pathogen Campylobacter jejuni has been solved at 1.65 A spacing. This essential bacterial enzyme is the second representative of the new family of dimeric dUTPases to be structurally characterised. Members of this family have a novel all-alpha fold and are unrelated to the all-beta dUTPases of the majority of organisms including eukaryotes such as humans, bacteria such as Escherichia coli, archaea like Methanococcus jannaschii and animal viruses. Therefore, dimeric dUTPases can be considered as candidate drug targets. The X-ray structure of the C.jejuni dUTPase in complex with the non-hydrolysable substrate analogue dUpNHp allows us to define the positions of three catalytically significant phosphate-binding magnesium ions and provides a starting point for a detailed understanding of the mechanism of dUTP/dUDP hydrolysis by dimeric dUTPases. Indeed, a water molecule present in the structure is ideally situated to act as the attacking nucleophile during hydrolysis. A comparison of the dUTPases from C.jejuni and Trypanosoma cruzi reveals a common fold with certain distinct features, both in the rigid and mobile domains as defined in the T.cruzi structure. Homologues of the C.jejuni dUTPase have been identified in several other bacteria and bacteriophages, including the dCTPase of phage T4. Sequence comparisons of these proteins define a new superfamily of d(C/U)TPases that includes three distinct enzyme families: (1) dUTPases in trypanosomatides, C.jejuni and several other Gram-negative bacteria, (2) predicted dUTPases in various Gram-positive bacteria and their phages, and (3) dCTP/dUTPases in enterobacterial T4-like phages. All these enzymes share a basic module that consists of two alpha-helices from the rigid domain, two helices from the mobile domain and connecting loops. These results in concert with a number of conserved residues responsible for interdomain cross-talk provide valuable insight towards rational drug design.     

Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.     

Longitudinal study of Campylobacter jejuni bacteriophages and their hosts from broiler chickens

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.     

Isolation and characterization of bacteriophages for avian pathogenic E. coli strains

Aims:  To isolate and characterize bacteriophages, and to evaluate its lytic performance against avian pathogenic Escherichia coli (APEC) strains with high patterns of antibiotic resistance, in order to select phages for a therapeutic product to treat colibacillosis in chickens.
Methods and Results:  Bacteriophages were isolated from poultry sewage and tested against 148 O-serotyped APEC strains. The morphological characterization of the bacteriophages was made by transmission electronic microscopy (TEM) observations and the genetic comparison between bacteriophages DNA was performed by restriction fragment length polymorphism (RFLP) patterns. Results showed that 70·5% of the tested E. coli strains were sensitive to a combination of three of the five isolated phages, that seemed to be virulent and taxonomically belong to the Caudovirales order. Two of them look like 16–19, T4-like phages ( Myoviridae ) and the third is a T1-like phage and belongs to Syphoviridae family. All of them are genetically different.
Conclusions:  It was possible to obtain a combination of three different lytic bacteriophages with broad lytic spectra against the most prevalent O-serotypes of APEC.
Significance and Impact of the Study:  Data reported in this study, presents an in vitro well studied phage product to be used as antimicrobial agent to treat colibacillosis in poultry industry.     

Reduction of Campylobacter jejuni colonization of chicks by cecum-colonizing bacteria producing anti-C. jejuni metabolites.

Cecum-colonizing bacteria were isolated from Campylobacter jejuni-free White Leghorn (Gallus domesticus) laying hens and screened for the ability to produce anti-C. jejuni metabolites. Nine isolates were obtained that possessed this characteristic. The peroral administration of the nine isolates as a mixture (ca. 10(9) per chick) to 1-day-old chicks was followed 1 week later by peroral inoculation of Campylobacter jejuni (ca. 10(9) per chick) to determine if the cecal isolates could protect chicks from colonization by campylobacters. The nine-strain mixture of cecal bacteria provided from 41 to 85% protection from C. jejuni colonization. The protective bacteria were reduced to a mixture of three strains on the basis of their ability to utilize mucin as a sole substrate for growth. These strains included Klebsiella pneumoniae 23, Citrobacter diversus 22, and Escherichia coli (O13:H-) 25. Four feeding trials with this three-strain mixture provided from 43 to 100% (average, 78%) protection from C. jejuni colonization. The dominant cecal bacterium of chicks treated with the three-strain mixture was consistently E. coli O13:H-. Similarly, three trials with only E. coli 25 used as the protective bacterium resulted in 49 to 72% (average, 59%) protection from C. jejuni colonization, with E. coli O13:H- being the dominant cecal bacterium in all cases. Although not completely effective, E. coli 25 substantially reduced the incidence of C. jejuni colonization of chicks. For all trials, fewer C. jejuni were present in the ceca of colonized chicks receiving the protective bacteria before exposure to C. jejuni than in chicks receiving only C. jejuni.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of Campylobacter jejuni colonization of chicks by cecum-colonizing bacteria producing anti-C. jejuni metabolites.

Cecum-colonizing bacteria were isolated from Campylobacter jejuni-free White Leghorn (Gallus domesticus) laying hens and screened for the ability to produce anti-C. jejuni metabolites. Nine isolates were obtained that possessed this characteristic. The peroral administration of the nine isolates as a mixture (ca. 10(9) per chick) to 1-day-old chicks was followed 1 week later by peroral inoculation of Campylobacter jejuni (ca. 10(9) per chick) to determine if the cecal isolates could protect chicks from colonization by campylobacters. The nine-strain mixture of cecal bacteria provided from 41 to 85% protection from C. jejuni colonization. The protective bacteria were reduced to a mixture of three strains on the basis of their ability to utilize mucin as a sole substrate for growth. These strains included Klebsiella pneumoniae 23, Citrobacter diversus 22, and Escherichia coli (O13:H-) 25. Four feeding trials with this three-strain mixture provided from 43 to 100% (average, 78%) protection from C. jejuni colonization. The dominant cecal bacterium of chicks treated with the three-strain mixture was consistently E. coli O13:H-. Similarly, three trials with only E. coli 25 used as the protective bacterium resulted in 49 to 72% (average, 59%) protection from C. jejuni colonization, with E. coli O13:H- being the dominant cecal bacterium in all cases. Although not completely effective, E. coli 25 substantially reduced the incidence of C. jejuni colonization of chicks. For all trials, fewer C. jejuni were present in the ceca of colonized chicks receiving the protective bacteria before exposure to C. jejuni than in chicks receiving only C. jejuni.(ABSTRACT TRUNCATED AT 250 WORDS)     

The chemical structure and genetic locus of Campylobacter jejuni CG8486 (serotype HS:4) capsular polysaccharide: the identification of 6-deoxy-D-ido-heptopyranose

In line with our on-going efforts to create a multivalent anti-Campylobacter jejuni vaccine based on its capsule polysaccharides (CPSs), we report here the chemical structure and the genetic locus of the CPS produced by C. jejuni strain CG8486, which belongs to the serotype HS:4 CPS complex. C. jejuni CG8486 CPS was observed to be composed of approximately 17 disaccharide repeating blocks of 4-substituted N-acetyl-beta-D-glucopyranosamine and 3-substituted 6-deoxy-beta-D-ido-heptopyranose. A small number of 6-deoxy-beta-D-ido-heptopyranose units were observed to carry O-methyl phosphoramidate moieties at the O-2 or O-7 position. The gene content and organization of the CPS locus of C. jejuni CG8486 were comparable to those of C. jejuni strains NCTC 11168 and 81-176, but several CG8486 CPS genes were observed to be more divergent from those present in the CPS loci of NCTC 11168 and 81-176 CPS, which indicated that there are genetic characteristics specific to the C. jejuni HS:4 CPS complex. The efficacy of a glycoconjugate vaccine based on C. jejuni CG8486 CPS is presently being tested in an animal model, the results of which will be presented in future communications.     

N-glycosylated proteins and distinct lipooligosaccharide glycoforms of Campylobacter jejuni target the human C-type lectin receptor MGL

An increasing number of bacterial pathogens produce an array of glycoproteins of unknown function. Here we report that Campylobacter jejuni proteins that are modified by the N -linked glycosylation machinery encoded by the pgl locus bind the human Macrophage Galactose-type lectin (MGL). MGL receptor binding was abrogated by EDTA and N -acetylgalactosamine (GalNAc) and was successfully transferred to Escherichia coli by introducing the C. jejuni pgl locus together with a glycan acceptor protein. In addition to glycoproteins, C. jejuni lipooligosaccharide with a terminal GalNAc residue was recognized by MGL. Recombinant E. coli expressing the C. jejuni pgl locus in the absence of a suitable glycan acceptor protein produced altered lipopolysaccharide glycoforms that gained MGL reactivity. Infection assays demonstrated high levels of GalNAc-dependent interaction of the recombinant E. coli with MGL-transfected mammalian cells. In addition, interleukin-6 production by human dendritic cells was enhanced by C. jejuni lacking N -linked glycans compared with wild-type bacteria. Collectively, our results provide evidence that both N -linked glycoproteins and distinct lipooligosaccharide glycoforms of C. jejuni are ligands for the human C-type lectin MGL and that the C. jejuni N -glycosylation machinery can be exploited to target recombinant bacteria to MGL-expressing eukaryotic cells.     

A specific DNA probe for the identification of Campylobacter jejuni

A 6.1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.     

Phagocytosis of Campylobacter jejuni and C. coli by peritoneal macrophages

Guinea-pig resident peritoneal macrophages had no activity against freshly isolated Campylobacter jejuni, whilst C. coli was phagocytosed and killed. The number of bacteria killed by macrophages always exceeded the number of those ingested, suggesting an extracellular mechanism of killing.     

Cell-surface alpha-glucan in Campylobacter jejuni 81-176

Campylobacter jejuni infection is a main source of severe gastroenteritis-related illnesses in humans and there is also evidence that it may be linked to neurological disorders. C. jejuni 81-176 is a virulent strain that has become the global model in the study of mechanisms and pathogenesis of C. jejuni infection. For this reason, we were engaged in studying the fine structures of cell-surface carbohydrate antigens of C. jejuni 81-176, namely, the capsule polysaccharide (CPS) and lipooligosaccharide (LOS). Serologically, C. jejuni 81-176 has been classified as belonging to serogroups HS23 and HS36, and indeed previous studies have shown that the LOS and CPS structures possess components similar to those expressed by serostrains HS23 and HS36. Here, we describe that in addition to the LOS and CPS, this strain also produced an independent cell-surface (1-->4)-alpha-glucan capsule.     

Application of bacteriophages in post-harvest control of human pathogenic and food spoiling bacteria

Bacteriophages have attracted great attention for application in food biopreservation. Lytic bacteriophages specific for human pathogenic bacteria can be isolated from natural sources such as animal feces or industrial wastes where the target bacteria inhabit. Lytic bacteriophages have been tested in different food systems for inactivation of main food-borne pathogens including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Salmonella enterica, Shigella spp., Campylobacter jejuni and Cronobacter sakazkii, and also for control of spoilage bacteria. Application of lytic bacteriophages could selectively control host populations of concern without interfering with the remaining food microbiota. Bacteriophages could also be applied for inactivation of bacteria attached to food contact surfaces or grown as biofilms. Bacteriophages may receive a generally recognized as safe status based on their lack of toxicity and other detrimental effects to human health. Phage preparations specific for L. monocytogenes, E. coli O157:H7 and S. enterica serotypes have been commercialized and approved for application in foods or as part of surface decontamination protocols. Phage endolysins have a broader host specificity compared to lytic bacteriophages. Cloned endolysins could be used as natural preservatives, singly or in combination with other antimicrobials such as bacteriocins.     

Campylobacter jejuni/coli in commercially reared beagles: prevalence and serotypes

A 5 year longitudinal study involving 187 commercially reared beagles from three suppliers was undertaken to determine prevalence and serotypes of Campylobacter jejuni and C. coli. Campylobacter jejuni or C. coli was isolated from the feces in 62 of 177 asymptomatic beagles and 8 of 10 dogs with diarrhea for an overall prevalence of 37%. A total of 36 isolates were serotyped on the basis of thermostable antigens with 20 antisera prepared against frequently occurring serotypes isolated from humans with campylobacter associated enteritis (15 C. jejuni, 5 C. coli serotypes). Of these isolates, 17 (47%) serotyped with antisera to 7 C. jejuni serotypes frequently isolated in human cases of enteric campylobacteriosis (serotypes 1, 4, 10, 16, 18, 19, 37). One C. coli reacted to antisera 24, 34, 37, one strain of C. coli to antisera type 37, and another C. coli to antisera type 34. All three C. coli belonged to serotypes frequently encountered in diarrheic human patients.     

噬菌体与宿主菌间的角逐

噬菌体和它们的宿主菌组成了地球上教目最庞大的微生物种群,噬菌体靠寄生宿主菌来扩增繁衍。但在漫长的进化中,噬菌体与宿主菌间不单是捕食关系,它们间还形成了复杂的相互对抗机制,其中抵抗防御机制的抗防御机制也会促使抗．抗防御机制的产生。从生态学角度来看,噬菌体和宿主菌间的共进化保持着动态平衡。本文综述近年来这一领域的研究,为更清楚地了解噬菌体与宿主菌间的关系和应用提供了参考。     

细菌与噬菌体相互抵抗机制研究进展

噬菌体作为一种侵染细菌的病毒,能够特异性识别宿主细菌。近年来,抗生素的过度使用导致耐药细菌的出现,噬菌体有望成为对抗耐药细菌的新武器。在细菌与噬菌体长期共进化过程中,二者都演化出一系列抵御策略。本文从抑制噬菌体吸附、阻止噬菌体DNA进入、切割噬菌体基因组、流产感染以及群体感应对噬菌体的调控等方面,对细菌抵抗噬菌体的机制以及噬菌体应对细菌的策略进行了综述,同时还列举了细菌和噬菌体相互抵抗机制的检测方法,以期为噬菌体在细菌控制中的应用以及探究细菌抵抗噬菌体的机制提供理论依据。     

Prevalence of campylobacters and arcobacters in ducks at the abattoir

Ten duck carcasses, five from each of two different flocks, and four pairs of pooled duck caecal contents, each pair from a separate flock, were examined by a variety of techniques for arcobacters and campylobacters. Campylobacter coli, C. jejuni ssp. jejuni , C. upsaliensis, Arcobacter cryaerophilus and A. butzleri were isolated from duck caecal contents. Campylobacter coli, C. jejuni ssp. jejuni, A. cryaerophilus, A. butzleri and A. skirrowii were isolated from carcasses. The most effective methods for isolating these bacteria from carcasses involved selective enrichment in campylobacter enrichment broth, containing a cefoperazone, amphotericin, teicoplanin supplement, followed by plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA), or plating onto non-selective blood agar after filtration through a 0·65 μm pore size cellulose acetate filter. In contrast, recovery from caecal contents was most effective by direct plating onto mCCDA. API test strips performed poorly, failing to identify A. skirrowii or A. butzleri (which are not included in the scheme), or even many common campylobacters. The Preston biochemical characterization scheme was more helpful, though it did not distinguish between Arcobacter species. The species of most isolates of campylobacter, identified using the Preston scheme, was confirmed by the use of SDS-PAGE of whole cell proteins and this technique was also used successfully to speciate arcobacters.     

Prevalence of Campylobacter spp. in skinless, boneless retail broiler meat from 2005 through 2011 in Alabama, USA

ABSTRACT: BACKGROUND: The prevalence of Campylobacter spp. in 755 skinless, boneless retail broiler meat samples (breast, tenderloins and thighs) collected from food stores in Alabama, USA, from 2005 through 2011 was examined. Campylobacter spp. were isolated using enrichment and plate media. Isolates were identified with multiplex PCR assays and typed with pulsed field gel electrophoresis (PFGE). Data were analyzed by nominal variables (brand, plant, product, season, state and store) that may affect the prevalence of these bacteria. RESULTS: The average prevalence of Campylobacter spp. in retail broiler meat for these years was 41%, with no statistical differences in the prevalence by year (P > 0.05). Seasons did not affect the prevalence of C. jejuni but statistically affected the prevalence of C. coli (P < 0.05). The prevalence by brand, plant, product, state and store were different (P < 0.05). Establishments from two states had the highest prevalence (P < 0.05). C. coli and C. jejuni had an average prevalence of 28% and 66%, respectively. The prevalence of C. coli varied by brand, plant, season, state, store and year, while the prevalence of C. jejuni varied by brand, product, state and store. Tenderloins had a lower prevalence of Campylobacter spp. than breasts and thighs (P < 0.05). Although no statistical differences (P > 0.05) were observed in the prevalence of C. jejuni by season, the lowest prevalence of C. coli was recorded from October through March. A large diversity of PFGE profiles was found for C. jejuni, with some profiles from the same processing plants reappearing throughout the years. CONCLUSIONS: The prevalence of Campylobacter spp. did not change during the seven years of the study; however, it did change when analyzed by brand, product and state. Seasons did not affect the prevalence of C. jejuni, but they did affect the prevalence of C. coli. Larger PFGE databases are needed to assess the temporal reoccurrence of PFGE profiles to help predict the risk associated with each profile.     

The heterogeneity of Campylobacter jejuni and Campylobacter coli strains isolated from healthy cattle

AIMS: To identify campylobacters isolated from clinically healthy cattle at species level by a multiplex polymerase chain reaction (m-PCR). The heterogeneity among Campylobacter jejuni and Campylobacter coli isolates was also investigated by using a restriction fragment length polymorphism (RFLP) analysis of flagellin (flaA) gene. METHODS AND RESULTS: Samples of intestinal contents, gall bladders, liver and faeces were collected from a total number of 1154 healthy cattle. The samples were inoculated onto Preston enrichment broth and agar. Of 1154 samples, 301 (26.1%) were positive for Campylobacter spp. Using an m-PCR assay for species identification, 179 (59.5%) were positive with C. jejuni specific primers while 30 (10%) were positive with C. coli specific primers. None of the liver samples examined was positive for C. jejuni or C. coli by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of the 209 isolates tested, 28 different flaA types were found. Twenty-three flaA types were identified among 179 C. jejuni isolates and the remaining five from C. coli isolates. CONCLUSIONS: Although the overall results suggest that the degree of heterogeneity among the flaA genes of thermophilic Campylobacter strains isolated from healthy cattle is relatively high, they should be treated cautiously as the number of band types for C. coli was low and band type 8 in C. jejuni was represented by a high percentage (%58). SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that healthy cattle can play role in the contamination of environment and human food chain by Campylobacter spp.     

Characterization of two bacteriophages specific to Acinetobacter baumannii and their effects on catheters biofilm

Multidrug-resistant strains of Acinetobacter baumannii cause major nosocomial infections. Bacteriophages that are specific to the bacterial species and destroy bacteria can be effectively used for treatment. In this study, we characterized lytic bacteriophages specific to A. baumannii strains. We isolated lytic bacteriophages from environmental water samples and then investigated their morphology, host range, growth characteristics, stability, genome analysis, and biofilm destruction on the catheter surface. Our results showed that the efficacy of the phages varied between 32% and 78%, tested on 78 isolates of A. baumannii; 80 phages were isolated, and two lytic bacteriophages, vB_AbaP_HB01 (henceforth called C2 phage) and vB_AbaM_HB02 (henceforth called K3 phage), were selected for characterization. Electron microscopy scans revealed that the C2 and K3 phages were members of the Podoviridae and Myoviridae families, respectively. Whole-genome sequencing revealed that the sequence of the C2 phage is available in the NCBI database (accession number: OP917929.1), and it was found sequence identity with Acinetobacter phage AB1 18%, the K3 phage DNA sequence is closely related to Acinetobacter phage vB_AbaM_phiAbaA1 (94% similarity). The cocktail of C2 and K3 phages demonstrated a promising decrease in the bacterial cell counts of the biofilm after 4 h. Under a scanning electron microscope, the cocktail treatment destructed the biofilm on the catheter. We propose that the phage cocktail could be a strong alternative to antibiotics to control the A. baumannii biofilm in catheter infections.