Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylobacter jejuni and Campylobacter coli.

Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviously, detecting reagents such as a specific antibody is essential for the development of immune-based detection methods for C. jejuni or C. coli. In this study, in silico techniques were used to design a chimeric recombinant antigen, named multiepitope antigen (MEA), for the production of specific polyclonal antibody. To design MEA polypeptide based on C. jejuni fibronectin-binding protein or CadF, four conserved and unique antigenic peptides were identified and fused together directly. The C. jejuni CadF-based MEA polypeptide fused with two single six-histidine tags at both C- and N-terminal ends was expressed under Escherichia coli expression system. The recombinant MEA was successfully produced and purified by Ni-NTA resin with a high satisfactory yield. Indirect ELISA results showed that anti-MEA polyclonal antibody derived from rabbit serum had a titer of 16,000, indicating high antigenicity of MEA polypeptide. Dot blot results also confirmed that the produced anti-MEA antibody could specifically recognize both C. jejuni and C. coli whole cells as expected while there was no cross-reactivity to non-Campylobacter spp. tested in this study.

Role of capsular polysaccharides and lipooligosaccharides in Campylobacter surface properties, autoagglutination, and attachment to abiotic surfaces.

The role of capsular polysaccharides and lipooligosaccharides in cell surface hydrophobicity, surface charge, autoagglutination (AAG), and attachment to abiotic surfaces of three strains of Campylobacter jejuni and one strain of C. coli were investigated. This was achieved by removal of capsular polysaccharides and truncation of lipooligosaccharides core oligosaccharides by inactivation of the kpsE and waaF genes, respectively. The mutants and the wild-type strains were compared after growth under planktonic (broth) and sessile (agar) conditions. Cells grown as planktonic cultures showed a significantly (p<0.05) higher degree of hydrophobicity and AAG activity but differed from their sessile counterparts with respect to surface charge and attachment counts, depending on the strain. These results suggest that prior mode of growth affects the surface properties and attachment of Campylobacter in a strain-dependent manner. There were no significant (p>0.05) differences between the three C. jejuni strains and their ΔkpsE and ΔwaaF mutants with respect to all traits tested. Inactivation of the kpsE gene significantly (p<0.05) reduced the surface charge of the C. coli strain from ∼-10 to ∼-6 mV and increased its AAG activity, while disruption of the waaF gene significantly (p<0.05) increased its surface hydrophobicity by >8° and decreased the numbers of cells attaching to stainless steel and glass by ∼0.5 log/cm². These results suggest that surface polysaccharides may influence the surface properties and attachment to abiotic surfaces of C. coli but not C. jejuni. This suggestion, however, requires further investigation using a larger number of strains of both species.

Evidence for phenotypic plasticity among multihost Campylobacter jejuni and C. coli lineages, obtained using ribosomal multilocus sequence typing and Raman spectroscopy.

Closely related bacterial isolates can display divergent phenotypes. This can limit the usefulness of phylogenetic studies for understanding bacterial ecology and evolution. Here, we compare phenotyping based on Raman spectrometric analysis of cellular composition to phylogenetic classification by ribosomal multilocus sequence typing (rMLST) in 108 isolates of the zoonotic pathogens Campylobacter jejuni and C. coli. Automatic relevance determination (ARD) was used to identify informative peaks in the Raman spectra that could be used to distinguish strains in taxonomic and host source groups (species, clade, clonal complex, and isolate source/host). Phenotypic characterization based on Raman spectra showed a degree of agreement with genotypic classification using rMLST, with segregation accuracy between species (83.95%), clade (in C. coli, 98.41%), and, to some extent, clonal complex (86.89% C. jejuni ST-21 and ST-45 complexes) being achieved. This confirmed the utility of Raman spectroscopy for lineage classification and the correlation between genotypic and phenotypic classification. In parallel analysis, relatively distantly related isolates (different clonal complexes) were assigned the correct host origin irrespective of the clonal origin (74.07 to 96.97% accuracy) based upon different Raman peaks. This suggests that the phenotypic characteristics, from which the phenotypic signal is derived, are not fixed by clonal descent but are influenced by the host environment and change as strains move between hosts.

Ribosomal operon intergenic sequence region (ISR) heterogeneity in Campylobacter coli and Campylobacter jejuni.

AIMS: The intergenic sequence regions (ISR) between the 16S and 23S genes of Campylobacter jejuni and Campylobacter coli are markedly different for each species. However, in the genomic sequence for Camp. coli RM2228, two rRNA operons have an ISR that is characteristic of Camp. coli, and the third operon is characteristic of Camp. jejuni. The aim of this study was to determine the prevalence of ISR heterogeneity in these organisms. METHODS AND RESULTS: PCR primers were designed to yield a 327-base pair (bp) product for Camp. coli and 166-bp product for Camp. jejuni. A strain like Camp. coli RM2228 should yield products of both sizes. DNA from a panel of Camp. coli (n=133) and Camp. jejuni (n=134) isolates were tested. All of the isolates yielded products of the predicted size for the species. To verify the data for Camp. coli RM2228, each ribosomal operon from the isolate was individually amplified by PCR and tested with the ISR primer pair. Products of both sizes were produced as predicted. CONCLUSIONS: The cross-species heterogeneity of the ISR seen in Camp. coli RM2228 is uncommon. SIGNIFICANCE AND IMPACT OF THE STUDY: The heterogeneity must have been caused by horizontal gene transfer at a frequency lower than predicted from housekeeping gene data. Thus, it can be expected that species identification based on the ISR can be confused in rare isolates.

Heat resistance and mechanism of heat inactivation in thermophilic campylobacters.

The heat resistance of Campylobacter jejuni strains AR6 and L51 and the heat resistance of Campylobacter coli strains DR4 and L6 were measured over the temperature range from 50 to 60 degrees C by two methods. Isothermal measurements yielded D55 values in the range from 4.6 to 6.6 min and z values in the range from 5.5 to 6.3 degrees C. Dynamic measurements using differential scanning calorimetry (DSC) during heating at a rate of 10 degrees C/min yielded D55 values of 2.5 min and 3.4 min and z values of 6.3 degrees C and 6.5 degrees C for AR6 and DR4, respectively. Both dynamic and isothermal methods yielded mean D55 values that were substantially greater than those reported previously (0.75 to 0.95 min). DSC analysis of each strain during heating at a rate of 10 degrees C/min yielded a complex series of overlapping endothermic peaks, which were assigned to cell wall lipids, ribosomes, and DNA. Measurement of the decline in the numbers of CFU in calorimetric samples as they were heated showed that the maximum rate of cell death occurred at 56 to 57 degrees C, which is close to the value predicted mathematically from the isothermal measurements of D and z (61 degrees C). Both estimates were very close to the peak m1 values, 60 to 62 degrees C, which were tentatively identified with unfolding of the 30S ribosome subunit, showing that cell death in C. jejuni and C. coli coincided with unfolding of the most thermally labile regions of the ribosome. Other measurements indicated that several essential proteins, including the alpha and beta subunits of RNA polymerase, might also unfold at the same time and contribute to cell death.

Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. "Species-identifying" biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within +/-5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) "strains" composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.

Phenotypic characterisation of flagellin and flagella of urease-positive thermophilic campylobacters.

In this study, flagellin is purified biochemically from eight urease-positive thermophilic camplylobacters (UPTC) isolated from river water, sea water and mussels, and purified also from two isolates of Campylobacter jejuni and C. coli and fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that no flagellin components were detected in the two Japanese UPTC isolates (CF89-12 and CF89-14) and the two UPTC NCTC strains (NCTC12893 and NCTC12894). Flagellin components, each consisting of a single peptide, with a heterogeneous molecular mass of approximately 52-63 kDa were demonstrated in the other four UPTC isolates (NCTC12892, NCTC12895, NCTC12896 and NI15F [from Northern Ireland]) and the two Japanese isolates of C. jejuni (JCM2013 and C. coli 27). The approximate molecular mass of flagellin from the flagellin-positive UPTC isolates was smaller than those of C. jejuni and C. coli. Flagella were not detected by electron microscopy in the four flagellin-negative UPTC isolates but they were detected in the four flagellin-positive UPTC isolates and the two isolates of C. jejuni and C. coli. Thus, significant phenotypic diversity for flagellin, which must be due to genotypic variations, was demonstrated among the UPTC isolates.

Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene.

Flagellins from Campylobacter jejuni 81-176 and Campylobacter coli VC167 are heavily glycosylated. The major modifications on both flagellins are pseudaminic acid (Pse5Ac7Ac), a nine carbon sugar that is similar to sialic acid, and an acetamidino-substituted analogue of pseudaminic acid (PseAm). Previous data have indicated that PseAm is synthesized via Pse5Ac7Ac in C. jejuni 81-176, but that the two sugars are synthesized using independent pathways in C. coli VC167. The Cj1293 gene of C. jejuni encodes a putative UDP-GlcNAc C6-dehydratase/C4-reductase that is similar to a protein required for glycosylation of Caulobacter crescentus flagellin. The Cj1293 gene is expressed either under the control of a sigma 54 promoter that overlaps the coding region of Cj1292 or as a polycistronic message under the control of a sigma 70 promoter upstream of Cj1292. A mutant in gene Cj1293 in C. jejuni 81-176 was non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. This mutant was complemented in trans with the homologous C. jejuni gene, as well as the Helicobacter pylori homologue, HP0840, which has been shown to encode a protein with UDP-GlcNAc C6-dehydratase/C4-reductase activity. Mutation of Cj1293 in C. coli VC167 resulted in a fully motile strain that synthesized a flagella filament composed of flagellin in which Pse5Ac7Ac was replaced by PseAm. The filament from the C. coli Cj1293 mutant displayed increased solubility in SDS compared with the wild-type filament. A double mutant in C. coli VC167, defective in both Cj1293 and ptmD, encoding part of the independent PseAm pathway, was also non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. Collectively, the data indicate that Cj1293 is essential for Pse5Ac7Ac biosynthesis from UDP-GlcNAc, and that glycosylation is required for flagella biogenesis in campylobacters.

Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins.

Flagellin from Campylobacter coli VC167 is post-translationally modified at > or = 16 amino acid residues with pseudaminic acid and three related derivatives. The predominant modification was 5,7-diacetamido-3,5,7,9 - tetradeoxy - l - glycero - l - manno - nonulosonic acid (pseudaminic acid, Pse5Ac7Ac), a modification that has been described previously on flagellin from Campylobacter jejuni 81-176. VC167 lacked two modi-fications present in 81-176 and instead had two unique modifications of masses 431 and 432 Da. Flagellins from both C. jejuni 81-176 and C. coli VC167 were also modified with an acetamidino form of pseudaminic acid (PseAm), but tandem mass spectrometry indicated that the structure of PseAm differed in the two strains. Synthesis of PseAm in C. coli VC167 requires a minimum of six ptm genes. In contrast, PseAm is synthesized in C. jejuni 81-176 via an alternative pathway using the product of the pseA gene. Mutation of the ptm genes in C. coli VC167 can be detected by changes in apparent Mr of flagellin in SDS-PAGE gels, changes in isoelectric focusing (IEF) patterns and loss of immunoreactivity with antiserum LAH2. These changes corresponded to loss of both 315 Da and 431 Da modifications from flagellin. Complementation of the VC167 ptm mutants with the 81-176 pseA gene in trans resulted in flagellins containing both 315 and 431 Da modifications, but these flagellins remained unreactive in LAH2 antibody, suggesting that the unique form of PseAm encoded by the ptm genes contributes to the serospecificity of the flagellar filament.

[Campylobacter jejuni and C. coli in aborted swine: comparison between phenotypic identification and polyacrylamide gel protein profiles]. / Campylobacter jejuni y C. coli en suinos abortados: comparación entre la identificación fenotípica y los perfiles proteicos en geles de poliacrilamida.

Campylobacter jejuni and Campylobacter coli were isolated from aborted pig fetuses which proceeded from different animals and farms between February 2000 and March 2001. Seven Campylobacter jejuni biotype II, three biotype I and one Campylobacter coli biotype I were identified by phenotypic tests and Lior's scheme. To corroborate and compare the phenotypic results, 7.5, 10 and 12.5% polyacrilamide gel electrophoresis (SDS-PAGE) were used under reducing conditions. Characteristic bands of hypervariable dense zone within C. jejuni and C. coli species were observed in all the whole cell protein extracts with differences in mobility. It was possible to establish differences between identical phenotypic Campylobacter isolates and different protein profile from fetuses of the same litter. SDS-PAGE is a stable and reproducible method to establish differences between Campylobacter strains and is considered applicable for the differentiation of the wide variability of Campylobacter species for epidemiologic purposes.

Detection of thermophilic Campylobacter from sparrows by multiplex PCR: the role of sparrows as a source of contamination of broilers with Campylobacter.

The best combination of primers and the annealing temperature of multiplex PCR for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari were examined. The multiplex PCR was able to detect type strains of the three species. All results of identification of wild strains (30 strains of C. jejuni, 20 strains of C. coli, and 4 strains of C. lari) by the multiplex PCR coincided with those of the conventional biochemical identification tests, suggesting that the multiplex PCR can simultaneously differentiate C. jejuni, C. coli, and C. lari from wild strains of campylobacters easily and rapidly. Campylobacters were detected from sparrow feces by the multiplex PCR and antimicrobial sensitivities of the strains were determined to discuss the role of sparrows in contamination of broilers with C. jejuni. Three out of 13 strains of C. jejuni isolated from sparrow feces showed quinolone resistance. From the frequent use of quinolones for treatment of industrial animals like chickens, pigs, and cows, the three strains of quinolone-resistant C. jejuni in sparrows must have been originated from those industrial animals. Sparrows that have quinolone-resistant C. jejuni were considered to have contacted with industrial animals or thier feed. It may be presumed, on the contrary, that C. jejuni in sparrows could be a potential source of contamination of broilers.

Characterization and gene sequencing of a 19-kDa periplasmic protein of Campylobacter jejuni/coli.

In order to study a 19-kDa protein (p19) of Campylobacter jejuni, we purified this protein to homogeneity from C. jejuni strain 81,176 by anion exchange chromatography. The molecular weight of the native protein is 19,000 daltons. P19 was found to be acidic with an isoelectric point of 4.8 and was located in the periplasmic space of the bacteria. The 20 N-terminal amino acids were sequenced and no significant similarities with known proteins were shown. A monoclonal antibody showed that p19 is conserved in the 2 species C. jejuni and C. coli. Analysis of sera from 23 patients with a Campylobacter-related infection indicated that p19 is not immunogenic during natural infection in man. The gene encoding p19 was cloned and no strong homologies with known sequences were identified. The preparation of a knockout mutant in p19 will enable the investigation of the function of this cell wall component of Campylobacter.

Characterization of a post-translational modification of Campylobacter flagellin: identification of a sero-specific glycosyl moiety.

The flagellins of Campylobacter spp. differ antigenically. In variants of C. coli strain VC167, two antigenic flagellin types determined by sero-specific antibodies have been described (termed T1 and T2). Post-translational modification has been suggested to be responsible for T1 and T2 epitopes, and, using mild periodate treatment and biotin hydrazide labelling, flagellin from both VC167-T1 and T2 were shown to be glycosylated. Glycosylation was also shown to be present on other Campylobacter flagellins. The ability to label all Campylobacter flagellins examined with the lectin LFA demonstrated the presence of a terminal sialic acid moiety. Furthermore, mild periodate treatment of the flagellins of VC167 eliminated reactivity with T1 and T2 specific antibodies LAH1 and LAH2, respectively, and LFA could also compete with LAH1 and LAH2 antibodies for binding to their respective flagellins. These data implicate terminal sialic acid as part of the LAH strain-specific epitopes. However, using mutants in genes affecting LAH serorecognition of flagellin it was demonstrated that sialic acid alone is not the LAH epitope. Rather, the epitope(s) is complex, probably involving multiple glycosyl and/or amino acid residues.

Effect of the complement system on the sensitivity of Campylobacter jejuni and Campylobacter coli to human blood serum.

We determined the sensitivity of five strains of Campylobacter jejuni and C. coli isolated from children with diarrhea and from chicken feces to normal human blood serum (undiluted and at concentrations of 10, 30, 50 and 70%), a hypogammaglobulinemic serum and a complement-deficient serum. Both species were highly sensitive to the bactericidal activity of human serum, regardless of their source. The highest bactericidal activity was observed with pooled fresh normal serum, with bacterial survival rates inversely correlated to serum dilutions. Inactivated serum had the least bactericidal activity. When complement was partially restored to inactivated serum, lower survival rates were observed. The hypogammaglobulinemic-normal complement-containing serum had strong bactericidal activity whereas the normal immunoglobulin-containing but complement-deficient serum had little bactericidal activity. These results suggest that Campylobacter may be able to directly activate complement by the alternative pathway.

Effect of the complement system on the sensitivity of Campylobacter jejuni and Campylobacter coli to human blood serum

We determined the sensitivity of five strains Compylobacter jejuni and C. coli isolated from children with diarrhea and from chicken feces to normal human blood serum (undiluted and at concentrationss of 10, 30, 50 and 70 per cent), hypogmmaglobulinemic serum and a complemented-deficient serum. Both species were highly sensitive to the bactecidal activity of human serum, regardless of their source. The highest bactericidal activity was observed with pooled fresh normal serum, with bacterial survival rates inversely correlated to serum dilutions. Inactivated serum had the least bactericidal activity. When complement was partially restored to inactivated serum, lower survival rates were observed. The hypogammaglobulinemic-normal complement-containing serum had strong bactericidal activity whereas the normal immunoglobulin-containing but complement-feficient serum had little bactericidal activity. These results suggest that Campylobacter may be able to directly activate complement by the alternative pathway

Structural and antigenic characteristics of Campylobacter coli FlaA flagellin.

The polar flagellar filament of Campylobacter coli VC167 is composed of two highly related (98%) flagellin subunit proteins, FlaA and FlaB, whose antigenic specificities result from posttranslational modification. FlaA is the predominant flagellin species, and mutants expressing only FlaA form a full-length flagellar filament. Although the deduced M(r) of type 2 (T2) FlaA is 58,884 and the apparent M(r) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 59,500, the solution weight-average M(r) by sedimentation analysis was 63,000. Circular dichroism studies in the presence or absence of 0.1% sodium dodecyl sulfate or 50% trifluorethanol showed that the secondary structure of T2 FlaA flagellin was altered, with alpha-helix structure being increased to 25% in the nonpolar environment. The molecule also contained 35 to 48% beta-sheet and 11 to 29% beta-turn structure. Mimeotope analysis of octapeptides representing the sequence of FlaA together with immunoelectron microscopy and enzyme-linked immunosorbent assay with a panel of antisera indicated that many residues in presumed linear epitopes were inaccessible or nonepitopic in the assembled filament, with the majority being in the N-terminal 337 residues of the 572-residue flagellin. Residues at the carboxy-terminal end of the T2 FlaA subunit also become inaccessible upon assembly. Digestion with trypsin, chymotrypsin, and endoproteinase Glu-C revealed a protease-resistant domain with an approximate M(r) of 18,700 between residues 193 and 375. Digestion with endoproteinase Arg-C and endoproteinase Lys-C allowed the mapping of a segment of surface-exposed FlaA sequence which contributes serospecificity to the VC167 T2 flagellar filament at residues between 421 and 480.

[The fatty acid composition of the total lipids in bacteria of the genus Campylobacter]. / Zhirnokislotnyi sostav summarnykh lipidov bakterii roda Campylobacter.

Composition of fatty acids of total lipids in home strains of campylobacteria has been studied. Lipids of all the strains of C. jejuni and C. coli mainly consist of saturated fatty acids (from 75.7 to 78.7%) with predominance of tetradecanoic and hexadecanoic fatty acids. The level of unsaturated fatty acids is considerably lower (from 21.0 to 22.5%); These acids are mainly presented by hexadecene acid. Qualitative composition of fatty acids of total lipids in C. jejuni and C. coli does not permit using it for differentiation within these species.

Lipopolysaccharide of Campylobacter coli serotype O:30. Fractionation and structure of liberated core oligosaccharide.

Lipopolysaccharide from phenol-water extraction of cells of Campylobacter coli serotype O:30 was separated as a water-insoluble gel of low M(r) from a water-soluble antigenic polysaccharide of high M(r). Acetic acid hydrolysis of the ketosidic linkages to lipid A in the lipopolysaccharide furnished a core oligosaccharide. Structural investigations were performed using 1H and 13C NMR, fast atom bombardment-mass spectrometry of permethylated derivatives, and methylation linkage analysis on the core oligosaccharide and the products of two successive Smith degradations. It is concluded that the highly branched 3-deoxy-D-manno-octulosonic acid-terminated oligosaccharide chains carried at the nonreducing end disaccharide units of beta-D-Qui3NAc-(1-->2)-beta-D-Qui3NAc (where Qui3NAc represents 3-acylamino-3,6-dideoxy-D-glucose), in which N-acyl residues were either both (R)-3-hydroxybutanoyl or both 3-hydroxy-2,3-dimethyl-5-oxoprolyl. The demonstration of these unusual features provides further evidence for a wide variety of structures within the core oligosaccharide region of lipopolysaccharides from Campylobacter sp.

Differentiation of thermophilic species of Campylobacter, in particular C. coli and C. jejuni, with atypical characteristics, by analysis of protein-banding profiles on non-denaturing polyacrylamide gels.

The protein-banding patterns after electrophoresis of 23 strains of thermophilic Campylobacter, including 17 strains of C. coli and C. jejuni which had atypical characteristics with respect to the hydrolysis of hippurate and susceptibility to nalidixic acid, were characterized. Of the atypical strains of C. coli and C. jejuni 16 out of 17 gave protein-banding patterns which were essentially identical to those of typical strains, and distinct patterns were obtained from strains of two other species. The banding patterns of soluble proteins on non-denaturing polyacrylamide gels after electrophoresis appear to be useful for the differentiation of atypical strains of C. coli and C. jejuni. Strain 11791 which was originally identified as C. coli was identified as C. lari from its protein-banding profile.

Characterization of viable but nonculturable stage of C. coli, characterized with respect to electron microscopic findings, whole cell protein and lipooligosaccharide (LOS) patterns.

Campylobacter coli CK 205, isolated from swine feces, was examined for changes in cell morphology, protein and lipooligosaccharide (LOS) patterns during starvation-survival experiments. Bottles filled with sterile filtered A. dest. were seeded with campylobacters and incubated at 4 degrees C and 37 degrees C. Transition to the nonculturable stage occurred within 48 hours (37 degrees C) and 2 weeks (4 degrees C), respectively. In contrast to the culturability the electrophoretic studies showed no changes in whole cell protein or LOS patterns. The electron microscopic pictures revealed spiral and coccoid forms, partly with a slightly enlarged periplasmatic space or budding of the membrane. Totally intact non culturable spiral or coccoid forms might be regarded as dormancy forms that cannot be detected by conventional microbiological methods in water examination.