Cloning and expression of the Campylobacter jejuni glyA gene in Escherichia coli.

Genetic studies of Campylobacter jejuni are greatly hampered by the lack of genetic markers and an established classical gene transfer mechanism between strains of this species. To facilitate future genetic studies and to provide a recombinant DNA approach for analyzing genes of C. jejuni, we constructed an extensive genomic library of a pathogenic C. jejuni strain TGH9011 (serotype 0:3) using pBR322. We report the isolation of a number of recombinant plasmids containing the complete structural gene of glyA, that encodes serine hydroxymethyltransferase (SHMT) of C. jejuni. Escherichia coli cells containing this multicopy recombinant plasmid with the glyA gene produce high levels of SHMT. The SHMT-encoding fragment was identified by subcloning and functional complementation. The expression of the C. jejuni glyA gene was probably via transcription initiated from its own promoter.

Pulmonary disposition of moxalactam.

Moxalactam is a new synthetic oxa-beta-lactam antibiotic with a broad spectrum of activity against Gram-positive and Gram-negative aerobic and anaerobic bacteria. It has proven clinical efficacy in pneumonia caused by a variety of infecting organisms. Therapeutic concentrations of moxalactam are achieved in most body tissues and fluids, including pleural fluid and sputum. However, assessment of the adequacy of lung tissue levels in pneumonia requires the sampling of material at an alveolar level. We performed bronchoalveolar lavage (BAL) in 13 patients one hour after they had been given moxalactam intravenously in doses ranging from 250 mg to 2 g. Absolute alveolar drug levels ranged from less than 1 to 6 micrograms/ml, and serum levels from 8 to 50 micrograms/ml. When expressed per micromole of creatinine, there was a significant relationship (r = 0.85; p less than 0.01) between serum and alveolar moxalactam levels in those patients in whom the drug concentration could be quantified accurately in BAL fluid.

Antigenic shifts in serotype determinants of Campylobacter coli are accompanied by changes in the chromosomal DNA restriction endonuclease digestion pattern.

Changes in somatic (O) lipopolysaccharide (LPS) antigenic specificities of Campylobacter coli serostrains were observed after continuous laboratory subculture. Two serostrains (C. coli O34 and C. coli O48) lost O specificity and did not react with homologous or any of the available heterologous antisera. The C. coli serostrain for serogroup O5, after subculture, yielded a variant that had acquired a new specificity which was detectable with a heterologous antiserum. In a repeat experiment with the original isolate of the O5 strain, a second variant was obtained which had not only acquired the same new determinant but had, unlike the first variant, lost reactivity with the homologous antiserum. Immunoblot experiments with homologous and heterologous antisera indicated that changes in antigenic specificity were associated with the O side chains of the LPS molecules. Results of restriction endonuclease analysis of chromosomal DNA of the variants and their parents revealed minor differences in restriction patterns which suggested that C. coli is capable of undergoing genomic re-arrangements that lead to changes in LPS specificity and structure.

Variation of the O antigen of Campylobacter jejuni in vivo.

During the course of a clinical study on patients with campylobacteriosis, three consecutive isolates of Campylobacter jejuni from the same patient were sent for O-serotyping. Marked differences in the specificities of the O antigens of the isolates were observed between the first and third isolates when a passive haemagglutination assay system developed for serotyping C. jejuni was used. Differences in specificity were also demonstrated by immunoblots of lipopolysaccharides (LPS) from proteinase K-digested whole cells. The three isolates could not be distinguished either by restriction endonuclease analysis of chromosomal DNA, by gel electrophoresis of whole-cell proteins or by silver-stained LPS gels, thus providing evidence that they were of the same strain and that antigenic variation had occurred in vivo.

Distribution of sero-biotypes of Campylobacter jejuni and C. coli isolated from paediatric patients.

During a one-year period, 258 isolates of Campylobacter jejuni and C. coli were obtained from children with gastroenteritis or bacteraemia at the Red Cross Children's Hospital, Cape Town, South Africa. These isolates were biotyped by hippurate hydrolysis, H2S production and tolerance to 2,3,5-triphenyltetrazolium chloride (TTC). Our study indicated that 95.4% of the isolates were C. jejuni biotype 1, 1.5% were C. jejuni biotype 2 and 3.1% were C. coli; 70% of the isolates were resistant to TTC. Serotyping on the basis of soluble, thermostable antigens detected by a passive-haemagglutination technique revealed that 79% of the Cape Town isolates were typable and that the most common serotypes, in order, were: 4, 2, 12, 23/36 and 19, together comprising 25% of the isolates. About 37% of the typable isolates belonged to nine serotypes. The finding that 21% of the isolates were non-typable suggests the existence of antigenic specificities different from those defined by the 60 antisera in current use.

Comparison of a blood-free medium and a filtration technique for the isolation of Campylobacter spp. from diarrhoeal stools of hospitalised patients in central Australia.

Single specimens of diarrhoeal stool from 676 patients, mostly aboriginals aged less than 5 years, admitted to Alice Springs Hospital, central Australia, for diarrhoea between Sept. 1988 and Feb. 1989, were examined for Campylobacter spp. by culture on a blood-free medium with selective supplement (BFM; Oxoid) and blood agar overlaid with a membrane filter (FM). Campylobacter spp. were isolated on either BFM or FM or both from 225 patients. Campylobacter spp. were isolated on BFM alone from 75 patients and on FM alone from 213 patients (p less than 0.001; chi 2 test). Most campylobacters isolated on BFM were C. jejuni. All C. jejuni subsp. doylei, all "C. upsaliensis" except one, all C. laridis, C. fetus subsp. fetus and several uncharacterised Campylobacter isolates were isolated on FM only. C. jejuni was isolated on BFM but not FM from several patients, and vice versa. Serotyping of C. jejuni and C. coli isolated from both media showed the serotypes recovered from the two media to be different in some patients. In some patients concurrent infection with several species or serotypes (up to five) of Campylobacter, or both, was shown for the first time by the use of FM. We conclude that the use in combination of a selective medium and a non-selective medium with a filtration technique are better than either medium alone for the isolation of Campylobacter spp.

Clinical and serological manifestations in patients during a waterborne epidemic due to Campylobacter jejuni.

A clinical and serological investigation of an epidemic due to Campylobacter jejuni in a community with a population of 1026 is presented. Altogether, 22 faecal samples from 27 patients were positive, with serotypes O 2 (n = 21) and O 6, 7 (n = 1) being identified. Serotype O 19, 21 was isolated from drinking water which had been consumed by 89.5% households answering a questionnaire, thereby indicating an attack rate of 66.5% (i.e. 680 persons). Mean duration of illness was 6.5 +/- 4.6 days. Diarrhoea (82.3%), abdominal pains (62.8%) and fever (41.8%) were the most common symptoms. Acute stage samples of serum from Campylobacter-positive patients had lower concentrations of IgG antibodies against the most common serotype (O 2) than against serotype O 6, 7 (P = 0.05), which had previously been implicated in epidemics in the region. More than 80% samples drawn after 1-2 weeks of illness were positive for either IgA, IgM or IgG antibodies to serotype O 2 with a dominance of IgA. In the convalescent group (n = 24), serum from only one patient who developed a long-lasting reactive arthritis had antibodies to all serotypes.

Lipo-oligosaccharide of Campylobacter lari strain PC 637. Structure of the liberated oligosaccharide and an associated extracellular polysaccharide.

Lipo-oligosaccharide from phenol-water extraction of cells of Campylobacter lari strain PC 637 was separated as a water-insoluble gel of low relative molecular mass (M(r)) from a water-soluble extracellular polysaccharide of high M(r). Structural investigations were performed on the lipo-oligosaccharide and the extracellular polysaccharide, variously using 1H, 13C, and 31P NMR spectroscopy, linkage analysis, and fast atom bombardment-mass spectrometry of permethylated derivatives of the glycans and their products of chemical and enzymic degradation. The following structures are proposed for the highly branched oligosaccharide region: [formula: see text] and for the tetraglycosyl phosphate repeating unit of the extracellular polysaccharide: [formula; see text]

Lipo-oligosaccharides of Campylobacter jejuni serotype O:10. Structures of core oligosaccharide regions from a bacterial isolate from a patient with the Miller-Fisher syndrome and from the serotype reference strain.

Lipo-oligosaccharide (LOSa) was obtained by phenol-water extraction of bacterial cells of an isolate PG 836, identified as Campylobacter jejuni serotype O:10, from a patient who subsequently developed the Miller-Fisher syndrome (MFS). The product was separated into a water-insoluble gel of low Mr and a water-soluble component of high Mr. The structure of the core oligosaccharide region in LOSa is reported herein for comparison with LOSb from the C. jejuni O:10 reference strain, and is based on investigations carried out on: (1) O-deacylated LOSa; (2) the core oligosaccharide (OS 1a) liberated on acetic acid hydrolysis of the ketosidic linkages to lipid A, with accompanying loss of N-acetylneuraminic acid residues; (3) the product of the removal of phosphate residues from OS 1a to give OS 2a; and (4) the Smith degradation of OS 2a to yield a mixture of Os 3a and OS 4a. The results revealed that the core oligosaccharide region in LOSa from the MFS bacterial isolate had chains (1a), of which some were terminated by an N-acetylneuraminobiose [Neu5Ac(alpha 2-8)Neu5Ac] unit in a GD3 [Neu5Ac-Neu5Ac-Gal] epitope, and the inner regions of which were different from those of other C. jejuni serotypes. Similar experiments on LOSb from bacterial cells of the C. jejuni O:10 reference strain showed that the core oligosaccharide unit [1a, R = P (phosphoric monoester)] of LOSa from the MFS isolate was more uniformly complete than that of the O:10 reference strain [1b, R = AEP (2-aminoethylphosphate)] differing in the nature of the phosphate substituent at the inner heptose residue. The close structural relationship of LOSa from the MFS associated bacterium to LOSb from the O:10 reference strain runs parallel to that of the previously studied Guillain-Barré syndrome (GBS) associated bacterium typed as C. jejuni O:19 in comparison with the lipo-oligosaccharide from the reference strain. Preliminary studies on the high Mr components showed that those from the O:10 strains were indistinguishable from each other, but were structurally unrelated to those from the GBS associated C. jejuni serotype O:19 isolates and the O:19 reference strain [G.O. Aspinall, A.G. McDonald, and H. Pang, Biochemistry, 33 (1994) 250-255].

Inhibition of pseudomonas strains in two soft contact lens soaking solutions.

Ten Pseudomonas aeruginosa and six Pseudomonas cepacia strains recently isolated from infected patients were tested for susceptibility to the antimicrobial activity of two commercially-available soft contact lens soaking solutions. No strain showed evidence of viability after two hours but survivors were found in both solutions after one-half hour and in one solution after one hour. The solution most effective in inhibiting the Pseudomonas strains contained chlorhexidine and disodium edetate but each solution was effective in reducing heavy inocula of both species within the times recommended by the manufacturers.

The genus Campylobacter: a decade of progress.

In 1977, microbiologists and clinicians were awakened to the importance of the genus Campylobacter when it was learned that one species, Campylobacter jejuni, was a major cause of human enteritis. In the following decade substantial advances were made in diagnosis, isolation technology, identification, classification, serotyping, and epidemiology. The genus has undergone rapid expansion as advantage was taken of the deoxyribonucleic acid-deoxyribonucleic acid hybridization technique in defining new species. The 14 species now included in the genus, however, constitute a widely diverse group, and one species, C. pylori, which is associated with human gastroduodenitis, is under consideration for reassignment to another genus. The nomenclature of the subspecies of C. fetus has been resolved and the role of C. fetus subsp. fetus as an agent of human infections has been more clearly defined. The thermophilic campylobacteria that are etiological agents of human enteritis now include three species, C. jejuni, C. coli, and C. laridis. Recently defined species that have also been implicated as enteritis-causing agents include C. hyointestinalis, "C. upsaliensis," "C. cinaedi," and "C. fennelliae." The aerotolerant campylobacteria are now included in the species C. cryaerophila, and the campylobacteria isolated from salt marshes are included in C. nitrofigilis. The taxonomy and nomenclature of C. sputorum have been revised. C. sputorum now consists of three biovars (biotypes). Two of these, biovar sputorum and biovar bubulus, were previously considered to be separate subspecies and the third, biovar fecalis, was previously regarded as a separate species and known as "C. fecalis." The former subspecies C. sputorum subsp. mucosalis has been elevated to the rank of species. C. mucosalis is metabolically closely related to C. consisus. Human pathogens have not been identified among C. sputorum, C. mucosalis, or C. concisus. The goal of this article is to review developments during the last 10 years with emphasis on changes in taxonomy that are important from the perspective of the clinical microbiologist.

Comparative activity of tobramycin and gentamicin against Pseudomonas, Proteus and Providencia species.

Tobramycin is a new antibiotic resembling gentamicin. We measured the minimal inhibitory concentrations of these two antibiotics against five bacterial species that cause hospital-acquired infections and are resistant to many presently available antibiotics. The organisms tested were 500 strains of Pseudomones aeruginosa, 100 strains of each of Proteus rettgeri and Pr. morganii, 50 strains of Pr. vulgaris and 250 strains of Providencia stuartii. Tobramycin was 2 to 4 times more active than gentamicin against Ps. aeruginosa; all except 6 of 70 strains resistant to 4 mug/ml of gentamicin were sensitive to 4 mug/ml of tobramycin. The two antibiotics showed a similar degree of activity against the other four species. Tobramycin promises to be of particular value in the treatment of Pseudomonas infections.

O antigen grouping of Morganella morganii (Proteus morganii) by slide agglutination.

Antisera were prepared against Morganella morganii (Proteus morganii) type strains from the scheme described by Rauss et al. (Acta Microbiol. Acad. Sci. Hung. 22:315--321, 1975) and Raus and Vörös (Acta Microbiol. Acad. Sci. Hung. 6:233--248, 1959; Acta Microbiol. Acad. Sci. Hung. 14:195--198, 1967). The specificities of the somatic (O) antigens were studied using the passive hemagglutination and slide agglutination techniques. Previously unreported interstrain relations were observed, and O groups were provisionally defined on the basis of related strains. Of 143 isolates, collected mostly from two hospitals, 96% could be placed in one or another of the O groups. Forty-eight percent belonged to O group 1 and most of these were of two serotypes O1ab, 2 (20%) and O1ad, 2 (12%).

Passive hemagglutination technique for serotyping Campylobacter fetus subsp. jejuni on the basis of soluble heat-stable antigens.

Antigenic materials were extracted from Campylobacter fetus subsp. jejuni strains by heating bacterial suspensions in saline at 100 degrees C and by exposure to ethylenediaminetetraacetic acid. The antigens were heat stable at 100 degrees C, capable of sensitizing sheep erythrocytes for agglutination in antisera, and able to elicit production of specific antibody in rabbits; they occurred with different immunological specificities in 23 strains. Antisera against the 23 strains could be used for discriminating among isolates of the species when the passive hemagglutination technique was used for serotyping. Three serotypes were more common than others among a collection of human isolates.

Differences among Providencia species in their in vitro susceptibilities to five antibiotics.

Significant differences among the three Providencia species (P. alcalifaciens, P. stuartii, and P. rettgeri) in antimicrobial susceptibilities to five antibiotics were shown. P. stuartii was the most resistant of the three species, and P. alcalifaciens was the most susceptible. P. rettgeri was similar to P. stuartii in susceptibilities to cefoxitin, cephalothin, and cefamandole but differed in showing greater susceptibilities to tobramycin and gentamicin. Cefoxitin (16 micrograms/ml) and cefamandole (16 micrograms/ml) inhibited a greater proportion of P. stuartii isolates than did cephalothin, tobramycin, or gentamicin. The susceptibilities of urea-positive isolates of P. stuartii resembled more closely the susceptibilities of urea-negative isolates of this species than those of P. rettgeri isolates, a finding consistent with the recent recommendation for transferring such urea-positive strains to P. stuartii. Among P. stuartii isolates, marked resistance to cefoxitin accompanied by susceptibility to cefamandole was predominantly restricted to isolates of one serotype (O55). The use of isolates that had been serotyped and classified according to recent proposals for taxonomic changes in the Proteeae provided for clearer demonstration of species differences in susceptibility.

Separate O-grouping schemes for serotyping clinical isolates of Proteus vulgaris and Proteus mirabilis.

Antisera were prepared against type strains of the original scheme of B. Perch (Acta Pathol. Microbiol. Scand. 25:703-714, 1948) and against newly defined types to produce separate schemes for O-grouping Proteus vulgaris and Proteus mirabilis. In assessing the schemes for their effectiveness it was found that 82% of 208 P. vulgaris isolates and 88% of 194 P. mirabilis isolates from two hospitals were typable. Only 3.4% of the P. vulgaris isolates agglutinated in P. mirabilis antisera, and 1.5% of the P. mirabilis agglutinated in P. vulgaris antisera, indicating that separation of the schemes would be more advantageous in routine typing. P. mirabilis of groups O3, O6, O10, O29, and O30 were most frequently isolated. Of the P. vulgaris isolates, 25% belonged to newly defined O-groups, and one of these was the largest with 14% of all isolates of this species. The application of serotyping using separate schemes for each species was advocated in epidemiological studies.

Application of O-serotyping in a study of Providencia rettgeri (Proteus rettgeri) isolated from human and nonhuman sources.

A somatic (O) antigen serotyping scheme for Providencia rettgeri (Proteus rettgeri) was modified to exclude O-type strains recently reclassified as urea-positive Providencia stuartii and was extended to include new serotypes to provide for serotyping on the basis of 93 O-antigens. Isolates from two hospitals, five public health laboratories, and nonhuman sources (polluted water and frogs) were serotyped. The 112 isolates collected from a large general hospital over a 99-month period were distributed among 42 O-serotypes. No serotype showed significant predominance that would suggest the occurrence of human strains that are more prone than others to cause human infections, but in an institution experiencing cross-infection, 11 of the 22 (50%) isolates belonged to one serotype. The 54 isolates from the five public health laboratories belonged to 33 serotypes, 15 of which were found also among hospital isolates. All but 5 of 99 frog isolates were typable, and the 94 typable isolates were separated into 25 serotypes. Each of the four isolates from polluted water samples was of a different serotype. Sixteen of the serotypes found in frogs and three found in water were also identified among human isolates.

Diversity of lipopolysaccharide structures in Campylobacter jejuni.

Immune blots of electrophoresed lipopolysaccharides extracted from 38 Campylobacter jejuni serostrains suggested the presence of O chains in 16 strains and their absence in 22. Structural analysis confirmed the presence of O chains in serostrains O:19, O:23, and O:36 and the absence of O chains in serostrains O:1, O:2, and O:3. The O:19 strain has O repeat units of beta-D-glucuronic acid amidated with 2-amino-2-deoxyglycerol and N-acetylglucosamine. The 0:36 O chain has four different but closely related repeat units that each consist of N-acetylglucosamine, galactose, and a heptose that is varied in structure from one repeat unit to the next. The O:23 O chain has three different repeat units identical to three of O:36. Except for O:3, core oligosaccharides of strains with or without O chains contain sialic acid (Neu5Ac) in the terminal regions that in many cases mimic the structures of human gangliosides. Three neuropathic isolates were found to have a core terminal trisaccharide (Neu5Ac alpha2-->8Neu5Ac alpha2-->3Galbeta1) that was not found in nonneuropathic strains.

Cross-protection of mice provided by active and passive immunization against experimental infections with virulent Proteus rettgeri and Providencia bacteria.

Immunization with Providencia and Proteus rettgeri Formalin-treated bacterial suspensions produced high levels of protection in mice against homologous and heterologous challenge. Mice were also cross-protected, but less effectively, by passive administration of rabbit type-specific antisera. The protective activity appeared to be due to an antigen common to strains of different O-serotypes. It was not detectable in agglutination reactions, and preliminary results indicate that it is thermostable, not being inactivated in its antibody binding capacity at 121 degrees C for 1 h.

Structural and antigenic properties of lipopolysaccharides from serotype reference strains of Campylobacter jejuni.

To investigate the molecular basis for heat-stable antigenic diversity in Campylobacter jejuni, lipopolysaccharides (LPSs) from serotype reference strains and serotyped isolates were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coupled with silver staining and immunoblotting. By silver staining, only low-Mr components, consisting of one major band and as many as three minor bands ranging in Mr from 4,500 to 5,000, were detected. However, by immunoblotting with homologous antisera, 10 of 34 strains were shown to have a series of high-Mr LPS components characteristic of molecules with O side chains of various lengths. Isolates of the same serotype as the reference strain that had high-Mr LPS molecules were also found to have high-Mr LPS and in one case of cross-reacting strains it was found that the cross-reaction was associated with antibodies against high-Mr LPS. The reactions of LPSs with homologous and heterologous antisera indicated that both high- and low-Mr-type LPSs were strain-specific antigens, but in some cases cross-reactions were noted. Evidently, all C. jejuni strains possess low-Mr LPS that is readily detectable by silver staining, but some serotypes also possess high-Mr LPS components that can be visualized by immunoblotting.