Species identification of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> ssp. <Emphasis Type="Italic">jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis> by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and PCR

The Campylobacter species strains (n = 42; isolated from clinical samples and deposited in Czech National Collection of Type Cultures, Prague) originally phenotypically (and biochemically) identified as Campylobacter jejuni were re-classified using molecular biological and mass spectrometric methods. Whole-cell MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) separated the isolates into two genetically related strains — C. jejuni (n = 26) and C. coli (n = 16) and, moreover, distinguished the intimate details in the group of tested strains. It also made it possible to create the MALDI-TOF MS dendrogram; similarly, the spectral characteristics were used for the 3D cluster analysis. Polymerase chain reaction (PCR) confirmed the results obtained by mass spectrometry. Both methods (PCR and MALDI-TOF MS) gave the same results which supports their suitability in the rapid and accurate Campylobacter-species determination. Part of this work was presented at the 24th Congress of Czechoslovak Society for Microbiology, Liberec (Czech Republic) 2007.     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

Comparison of Three Methods for Epidemiological Typing of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis>

A total of 168 Campylobacter strains (154 C. jejuni and 14 C. coli) isolated from human clinical samples and chicken meat were typed using Penner serotyping, randomly amplified polymorphic DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) with four restriction enzymes (Sac II, Sal I, Sma I, Kpn I).The 168 strains were found to represent 13 different Penner-types and 72 different RAPD-types. However, the discriminatory potential of PFGE was dependent on the restriction enzymes used. The 168 strains were divided into 74 (Sac II), 73 (Sal I), 72 (Sma I) and 69 (Kpn I) types. The DNA of some strains was not digested by Sal I, Sma I and Kpn I. Although three RAPD-types were further subdivided by PFGE, RAPD showed good discriminatory power and a high level of agreement with PFGE patterns in terms of strain differentiation.To compare the similarities of PFGE patterns (Sac II) among the strains, a dendrogram was constructed based on the unweighted pair group method with averages (UPGMA). In most cases, DNA types of C. coli were different from those of C. jejuni. The similarities between human and meat isolates were less than 0.42 except for one outbreak in which the isolates from both patients and chicken meat showed the same DNA types.     

The <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>/<Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic"> cjaA</Emphasis> (<Emphasis Type="Italic">cj0982c</Emphasis>) Gene Encodes an N-Glycosylated Lipoprotein Localized in the Inner Membrane

The Campylobacter coli 72Dz/92 cjaA gene (orthologue of cj0982c of C. jejuni NCTC 11168) product is a highly immunogenic, amino acid-binding protein. CjaA was palmitic acid-modified when processed in E. coli. In addition, site-directed mutagenesis of the Cys residue of the LAAC motif of its signal sequence confirmed that CjaA is a lipoprotein when processed in Campylobacter. Localization of the protein appeared to be host dependent. In Campylobacter, CjaA was recovered mainly as an inner-membrane protein, whereas in E. coli most of the protein was present in the periplasmic space. Interestingly, antiserum raised against Campylobacter glycine-extracted material also recognized CjaA produced by Campylobacter and Escherichia coli, indicating that at least part of the protein may be surface exposed. Site-directed mutagenesis of the Asn residues of two putative N-linked glycosylation sites (NIS and NFT) showed that CjaA is glycosylated and that only the first N-X-S/T sequeon serves as a glycan acceptor.     

Colonization of broilers by <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> internalized within <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis>

Although Campylobacter survives within amoeba in-vitro, it is unknown if intra-amoeba Campylobacter jejuni can colonize broilers. Five groups of 28 day-of-hatch chicks were placed into separate isolators. Groups (1) and (2) were challenged with page’s amoeba saline (PAS), and disinfected planktonic C. jejuni NCTC 11168, respectively. Groups (3), (4) and (5) were challenged with a C. jejuni positive control, C. jejuni in PAS, and intra-amoeba C. jejuni, respectively. After 1, 3, 7 and 14 days post challenge, seven birds from each unit were examined for C. jejuni colonization. For the first time we report that intra-amoeba C. jejuni colonized broilers.     

Temperature-dependent phenotypic variation of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> lipooligosaccharides

Background  

Campylobacter jejuni is a major bacterial cause of food-borne enteritis, and its lipooligosaccharide (LOS) plays an initiating role in the development of the autoimmune neuropathy, Guillain-Barré syndrome, by induction of anti-neural cross-reactive antibodies through ganglioside molecular mimicry.     

Rapid detection of virulence <Emphasis Type="Italic">stx</Emphasis>2 gene of Enterohemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> using two-step ultra-rapid real-time PCR

A rapid detection method for Enterohemorrhagic Escherichia coli (EHEC), which has the virulent stx2 gene, was developed using a two-step, ultra-rapid real-time (URRT) PCR. URRT PCR was designed to detect the stx2 gene using a microchip-based, real-time PCR system, GenSpector TMC-1000, which only has a 6 μl total reaction volume with an extremely short denaturation step and combined annealing/extension step (1 and 3 s, respectively) for each cycle. Specific primers for the stx2 gene were designed to amplify a 100 bp region known for genetic stability among the various EHEC strains. Using the URRT PCR method, stx2 gene could be detected in 7 min 8 s including melting point (Tm) analysis. The detection limit for the stx2 gene for URRT-PCR was estimated to be 3 c.f.u./PCR with the amplification product having a consistent Tm of 85.2 ± 0.4°C. This method was tested for the various applications relevant to the different EHEC strains and was useful for the rapid detection of stx2-carrying EHEC strains.     

Identification of nine sequence types of the 16S rRNA genes of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis> isolated from broilers

Background  

Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. Campylobacter jejuni is the Campylobacter sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of C. jejuni and C. coli and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of Sma I restricted genomic DNA of the strains.     

Development of an indirect competitive ELISA for detection of<Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp.<Emphasis Type="Italic">jejuni</Emphasis> O:23 in foods

An indirect enzyme immunoassay for rapid detection of Campylobacter jejuni subsp. jejuni 0:23 has been developed. Optimum concentrations of immobilized cells, polyclonal chicken IgY, and rabbit anti-IgY antibody-horseradish peroxidase conjugate were 3.1 CFU/nL, 10 microg/mL, and 8 microg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/microL, limit of quantification being 480 CFU/microL. By testing 5 chromogens, viz. 1,2-benzenediamine, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3',5,5'-tetramethylbenzidine, bi(4,4'-anisidine) and 3-methyl-2-benzothiazolinone hydrazone, in horseradish peroxidase substrate, 1,2-benzenediamine or 3,3',5,5'-tetramethylbenzidine as H-donors in the enzyme substrate provided the highest ELISA sensitivity. The applied polyclonal antibody was specific for homogeneous antigen. The cross-reactions were observed only with one strain of C. sputorum subsp. sputorum (21.5 %) and with G+ bacterium Micrococcus luteus (6.1 %). Preliminary tests have been performed with a limited number of artificially contaminated food samples. No matrix effects on the ELISA sensitivity were observed. The results (by means of ELISA) were comparable with those given by both a standard cultivation method performed according to CSN ISO 10272 and commercially available Singlepath Campylobacter GLISA-Rapid Test.     

Antimicrobial activity of copper surfaces against suspensions of <Emphasis Type="Italic">Salmonella enterica</Emphasis> and <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Salmonella enterica and Campylobacter jejuni are amongst the more prevalent bacterial pathogens that cause foodborne diseases. These microorganisms are common contaminants of poultry and poultry products. This study was aimed to evaluate the antibacterial activity of metallic copper surfaces on these important enteropathogens, and to determine the potential acquisition of copper by food exposed to this metal.     

Expression of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> butanol synthetic genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), β-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

Desulforubrerythrin from <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, a novel multidomain protein

A novel multidomain metalloprotein from Campylobacter jejuni was overexpressed in Escherichia coli, purified, and extensively characterized. This protein is isolated as a homotetramer of 24-kDa monomers. According to the amino acid sequence, each monomer was predicted to contain three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix bundle domain harboring a non-sulfur μ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV–vis, EPR, and resonance Raman spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein contains two FeCys₄ centers with reduction potentials of +240 mV (desulforedoxin-like center) and +185 mV (rubredoxin-like center). These centers are in the high-spin configuration in the as-isolated ferric form. The protein further accommodates a μ-oxo-bridged diiron site with reduction potentials of +270 and +235 mV for the two sequential redox transitions. The protein is rapidly reoxidized by hydrogen peroxide and has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 μmol H₂O₂ min⁻¹ mg⁻¹. Owing to its building blocks and its homology to the rubrerythrin family, the protein is named desulforubrerythrin. It represents a novel example of the large diversity of the organization of domains exhibited by this enzyme family.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Molecular analysis of type 3 fimbrial genes from <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Klebsiella</Emphasis> and <Emphasis Type="Italic">Citrobacter</Emphasis> species

Background  

Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Survival mechanisms and culturability of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> under stress conditions

Culture-based isolation and enumeration of bacterial human pathogens from environmental and human food samples has significant limitations. Many pathogens enter a viable but non-culturable (VBNC) state in response to stress, and cannot be detected via culturing methods. Favourable growth conditions with a source of energy and an ideal stoichiometric ratio of carbon to inorganic elements can reverse this VBNC state. This review will focus on the bacterium Campylobacter jejuni which is a leading cause of food borne illness in the developed world. C. jejuni can enter a VBNC state in response to extremes in: pH, moisture content, temperature, nutrient content and salinity. Once in a VBNC state, the organism must maintain an energy balance from substrate oxidation through respiration to grow, divide and remain viable. The goal of this review is a greater understanding of how abiotic stress and thermodynamics influence the viability of C. jejuni.     

Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sokawa et al. suggest that rel^- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.