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Variation amongst strains of Helicobacter pylori was examined by 35S-methionine-labelled protein SDS-PAGE (Radio-PAGE), immunoblot and DNA fingerprinting techniques. These methods allowed discrimination amongst isolates and showed total correlation. Pig and baboon gastric Helicobacter pylori-like organisms were found to be very similar to Helicobacter pylori by both Radio-PAGE and immunoblotting, whereas a Helicobacter mustelae isolate was markedly different. The HindIII restriction endonuclease digest patterns of Helicobacter pylori DNA illustrated the considerable genomic variation of this organism. The Radio-PAGE and immunoblot typing methods both gave precise identification of Helicobacter pylori strains, but Radio-PAGE was found to give higher resolution and represents a standardised universally applicable fingerprinting method for Helicobacter pylori.

The enzyme activities responsible for carboxylation reactions in cell extracts of the gastric pathogen Helicobacter pylori have been studied by H14CO3- fixation and spectrophotometric assays. Acetyl coenzyme A carboxylase (EC 6.4.1.2) and malic enzyme (EC 1.1.1.40) activities were detected, whereas pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.3.1) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activities were absent. However, a pyruvate-dependent, ATP-independent, and avidin-insensitive H14CO3- fixation activity, which was shown to be due to the isotope exchange reaction of pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1), was present. The purified enzyme is composed of four subunits of 47, 36, 24, and 14 kDa. N-terminal sequence analysis showed that this enzyme is related to a recently recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pylori was found to mediate the reduction of a number of artificial electron acceptors in addition to a flavodoxin isolated from H. pylori extracts, which is likely to be the in vivo electron acceptor. Indirect evidence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.

The importance of the outer membrane and its pathogenesis of Helicobacter pylori in gastroduodenal disease has not fully determined. Surface-exposed proteins or those that may be exported to the surface may play an important role in the organisms’ colonization, maintenance and pathogenic action on gastric epithelial cells. It has previously been reported that H. pylori possesses three major outer memb 31 kDa [6] and which have all been shown to be highly immunogenic [2]. Acid extractable preparations and 125 I surface labelling of H. pylori has demonstrated several variable proteins, including a 48-kDa antigen [6, 8]. Genotypic diversity, already described for this organism, may allow variation in antigentic determinants on the cell surface [7]. Such a phenomenon may not only explain the presence of variable proteins on the membrane but may play a role in assisting the organism in evading the host’s immune system. This led us to investigate membrane-associated proteins from H. pylori. The colning of genes encode these proteins in Escherichia coli should provide information not only on virulence, but both phenotypic and genotypic interstrain variation and possible regulatory sequences. We isolated a crude membrane prepration to which polyclonal antibody was prepared and used to screen a lambda (λ) EMBL 3 H. pylori genomic library.

In order to fully investigate the role of Helicobacter pylori in the aetiology and pathogenesis of peptic ulcer disease sensitive methods of identifying and fingerprinting isolates of this organism are required. Conventional typing methods for bacteria, such as phage typing and serotyping have proven largely ineffective in typing H. pylori. Limited success has been achieved using systems based upon preformed enzymes such as the API-Zym system; however, the biotypes defined by such systems are limited and a large proportion of isolates fall into one biotype [1]. The more successful methods of identifying and fingerprinting H. pylori make use of molecular biological methods such as sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), restriction endonuclease digestion of genomic DNA and ribosomal RNA gene hybridisation patterns [2, 3].

A simple and reliable technique was developed for differentiating Helicobacter pylori strains by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified DNAs. Oligonucleotide primer pairs developed to the urease, 48-kDa stress protein (htrA), and 26-kDa antigen-encoding genes were used to amplify fragments of the appropriate size from crude boiled cell preparations. The PCR-amplified products were digested with Sau3A, HaeIII, MspI, AluI, MluI, HinfI, and XbaI restriction endonucleases. Restriction fragment length polymorphisms were particularly evident within the urease and htrA genes and were easily detected by Sau3A, HaeIII, MspI, and AluI restriction endonuclease analysis. Double digestion of these separately amplified products or restriction analysis of multiple PCR-amplified fragments was found to discriminate 17 of 17 (100%) H. pylori strains which had unique genomic DNA fingerprints. Results of an investigation of multiple isolate sets obtained from patients before and after therapy was consistent with the hypothesis that treatment failures were due to the persistence of the same strain but did not discount the possibility that the patients were reinfected with a strain shared by family members or close contacts. The results indicate that the PCR-restriction endonuclease analysis method can be applied directly to biopsy samples, has the potential to fingerprint H. pylori isolates rapidly, and may permit detailed epidemiological investigations on the transmission of this important pathogen.

Western blots of whole-cell sonicates of 10 different clones of a faecal isolate of Campylobacter jejuni 533 detected the expression of flagella antigens of either 59 or 62 kDa. Other antigenic proteins appeared identical both in the parent and all the clones. The mechanism for this phenotypic variation was studied using Southern blotting with a flagellin-specific gene probe and products of a polymerase chain reaction (PCR) using flagellin-gene primers. Restriction-enzyme digestion and Southern blotting did not detect any genomic rearrangements in the flagellin genes of the different phenotypes nor did restriction-enzyme analysis of the PCR products.

Summary A 1.5kb cryptic plasmid was isolated from Helicobacter pylori. Low-stringency hybridization analysis using this plasmid as a DNA probe revealed base sequence homology with other plasmids in this species. Nucleotide sequence analysis identified an open reading frame encoding a putative polypeptide of 25 kDa. This protein showed marked amino acid sequence similarity to replication-initiation proteins commonly found in small plasmids endogenous to Gram-positive bacteria which replicate by the ‘rolling-circle’ mechanism. Sequence motifs corresponding to the origins-of-replication consensus sequences were found on this cryptic plasmid. DNA and oligonucleotide probes to these plasmid replication sequences were used in hybridization analysis to identify similar sequences in other H. pylori plasmids. We believe this is the first plasmid isolated from a Gram-negative bacterium to show replication determinants characteristic of the ‘rolling-circle’ group of plasmids from Gram-positive bacteria. The cloned plasmid will be used to develop a shuttle-vector for H. pylori.

A 4.5-kilobase PstI fragment encoding part of the toxin A gene was isolated and used as a DNA probe in colony hybridization studies with 58 toxigenic and 17 nontoxigenic Clostridium difficile strains. All 58 toxigenic strains showed positive hybridization, in contrast to the 17 nontoxigenic strains. Southern blot analysis with the toxin A gene probe showed hybridization to a single fragment of equal intensities for HindIII-digested genomic DNAs isolated from C. difficile strains of wide-ranging toxin production. The positive hybridization signals were due to fragments of heterogeneous lengths (9 to 13 kilobases) for toxigenic strains of different types but were absent for the nontoxigenic strains. These results suggest the presence of a single copy of the toxin A gene on the genome of C. difficile strains, and the wide variation of toxin expression is not a reflection of gene copy number. The lack of toxin activity for nontoxigenic strains can be explained by the absence of at least part of the toxin A gene. The toxin A gene probe was tested against clostridial strains from 18 other species, of which only toxigenic C. sordellii strains showed positive hybridization. The specificity of the toxin A gene probe for toxigenic strains may lead to improved methods for the specific identification of toxigenic C. difficile strains from clinical specimens.

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.

The genes encoding the two structural subunits of the Helicobacter(Campylobacter) pyloriurease enzyme were cloned and sequenced. The derived amino acid sequences of the two H. pylori urease genes showed marked homology to both jack bean and soybean ureases. Almost 57% of residues were identical in the bacterial and plant ureases. This degree of similarity between a eucaryotic protein and a bacterial protein is almost unprecedented. The close similarity of the bacterial protein to the plant enzymes is an evolutionary anomaly that is perhaps best explained by horizontal gene transfer. The polymerase chain reaction technique using oligonucleotide primers to the H. pylori urease DNA sequence was found to sensitively detect H. pylori cells and may allow determination of the source and route of infection of the organism.

Helicobacter (Campylobacter) pylori has been implicated in the etiology of gastritis and peptic ulcer disease. H. pylori infection is normally diagnosed by microscopy and culture of biopsy samples. However, this is not only invasive but is time consuming and expensive. Various noninvasive serological techniques have been developed for diagnosing infection and ELISA is the most commonly used test. ELISAs that are based on H. pylori whole cells, sonicates or crude extracts have levels of sensitivity and specificity of 70%–90% [4]. A slight improvement has generally been reported by using acid cell extracts [7]. However, all these preparations contain antigens that cross-react with other bacterial species and cause false-positive reactions [5]. Adjusting the cut-off value only increases the number of false negatives. Second-generation ELISA systems have been developed which are based on high molecular weight conserved H. pylori specific purified antigens [4, 5]. They have high sensitivity and specificity and contain urease as the major antigenic component. High molecular weight fractionated antigens of H. pylori have also been isolated from SDS-PAGE gels [6] and these preparations may also be used for a H. pylori specific ELISA system. Molecular cloning of H. pylori immunodominant polypeptides can provide a useful, quick, high yield of H. pylori antigens that can be standardised and used for a specific diagnostic ELISA. We have previously reported the cloning of genes encoding 66 and 31 kDa immunodominant polypeptides of H. pylori in E.coli [3]. These antigens were demonstrated to be conserved, H. pylori-specific subunits of the H. pylori urease enzyme [2].

A gene bank from a clinical isolate of Clostridium difficile expressing high chloramphenicol acetyltransferase activity was constructed by cloning Sau3A-cleaved clostridial DNA fragments into the plasmid vector pUC13. Among 1,020 clones tested, 11 were resistant to chloramphenicol; 1 of these, with an insert size of 1.9 kilobases (pPPM9), was studied further. The clone pPPM9 was mapped using a variety of restriction enzymes, and a 0.27-kilobase EcoRV-TaqI restriction fragment was shown to be within the chloramphenicol resistance (Cmr) gene by using transposon (Tn1000) mutagenesis. The 0.27-kilobase fragment and the 1.9-kilobase insert were radiolabeled and used as DNA probes in hybridization studies. Southern blot analysis with the gene probes against chromosomal DNA from Cmr strains of C. difficile obtained from five distinct geographical locations revealed that at least two copies of the same chloramphenicol acetyltransferase gene were present for each strain. Hybridization of the gene probes against Cmr strains of Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella edwardsii, Escherichia coli, and to four other clostridial species revealed no homology even under conditions of low stringency.

A simple physical model of the catheterised bladder has been used to assess the activities of antiseptic agents that have been recommended as bladder instillations in the treatment of urinary tract infections in patients with indwelling catheters. The activities of povidone-iodine, phenoxyethanol, chlorhexidine, chlorhexidine supplemented with EDTA + Tris, noxythiolin and neomycin were examined against selected species of uropathogens. Except for phenoxyethanol against Pv. stuartii and possibly Ps. aeruginosa, single applications of these antibacterials for 30 min were not effective in sterilising urine in the artificial bladder. However, a second application 1 h later of phenoxyethanol but not chlorhexidine or povidone-iodine did eradicate Ps. aeruginosa. It is suggested that bacteria growing as a film on the bladder wall are particularly resistant to antimicrobials.

R′ plasmids have been generated that contain a determinant of Shigella dysenteriae 1 which directs the synthesis of Shiga toxin in Escherichia coli K-12. This gene is closely linked to the argE locus and thus is located in a region of the Shigella chromosome known to contain determinants of virulence factors.

DNA probes to identify the genes encoding toxins in enteric bacteria have been developed. Use of these probes reduces the number of animals required for toxicity testing, as suspect bacteria can be directly tested for the presence of toxin. We have augmented the gene probes available by developing probes against the Escherichia coli enterotoxin LTII and shiga toxin from Shigella dysenteriae 1. The LTII gene from E. coli 357900 was identified and characterised and a suitable internal probe was obtained. The LTII gene was found not to be common among enterobacteriae from various geographical locations. Isolates predominately of animal origin from Nigeria and Thailand hybridized with the probe. The shiga toxin gene was isolated from S. dysenteriae 1 by a combination of in vivo and in vitro methods. An internal probe was identified and used against different serogroups of Shigella and E. coli isolates. The probe was found to hybridize with S. dysenteriae 1 isolates and also some S. flexneri and S. sonnei strains. Representatives were tested for toxin production and found to produce toxin at low levels.

Heat-labile enterotoxin (LT)-producing Escherichia coli strains, as identified by the Y1 adrenal cell assay, were examined with a DNA probe coding for type I and type II LTs. Of 236 LT-producing E. coli isolates, 60% hybridized with LT-I, 17% hybridized with LT-II, and 23% did not hybridize with either probe and no longer produced LT as determined by the Y1 adrenal cell assay. These isolates presumably lost plasmids coding for LT-I during storage. A total of 75% of LT-producing E. coli isolates (27 of 36) from cows, 64% of LT-producing E. coli isolates (7 of 11) from buffalo, 31% of LT-producing E. coli isolates (4 of 13) from beef obtained in markets, and 2% of LT-producing E. coli isolates (3 of 168) from humans contained genes coding for LT-II. Genes coding for LT-II were not found in 50 LT-I-producing and heat-stable enterotoxin-producing E. coli isolates from 11 children with diarrhea and 44 LT-nonproducing and heat-stable enterotoxin-producing E. coli isolates from 12 other children with diarrhea. A total of 9% of LT-II-producing E. coli isolates (3 of 34) from cows and buffalo hybridized with DNA probes for genes coding for verocytotoxin 2 (VT2), and 18% (6 of 34) hybridized with a DNA probe coding for enterohemorrhagic E. coli (EHEC) adhesin fimbriae. E. coli SA-53, the original isolate in which LT-II was found, contained genes coding for VT2 and EHEC adhesin fimbriae. Five VT-producing, LT-II-producing E. coli isolates that hybridized with the EHEC probe did not contain DNA sequences coding for VT1 or VT2. LT-II-producing E. coli strains were frequently isolated from cattle and buffalo but were rarely isolated from humans.

A gene bank of Campylobacter pylori DNA in Escherichia coli was constructed by cloning Sau3A-cleaved DNA fragments into the bacteriophage vector lambda EMBL3. The expression of C. pylori antigens was determined by screening the gene library with adsorbed C. pylori whole-cell rabbit antisera. One recombinant clone which reacted positively (lambda CP2) was studied further. Immunoblot analysis with lambda CP2 showed a polypeptide band of 66 kilodaltons (kDa) reacting antigenically with the adsorbed antiserum. Extraction of DNA from lambda CP2 and digestion with SalI revealed a DNA insert of 17 kilobases (kb). Subcloning with SalI and the E. coli vector pUC18 showed that the DNA also encoded a 31-kDa antigen. The cloned antigens were shown by immunoblotting to have the same molecular weight in E. coli as in C. pylori and to be present in all C. pylori strains. Antiserum was raised against the cloned polypeptides and found to react only with C. pylori when analyzed by dot blotting and indirect immunofluorescence. The cloned antigens were determined to be expressed from the pUC18 lac promoter. The DNA encoding these antigens was radiolabeled with 32P and found to hybridize only to C. pylori strains. Immunoblotting with affinity-purified polyclonal antibody to the urease enzyme of C. pylori revealed that the cloned antigens may be part of the urease enzyme.

Isolates of Providencia stuartii , Pseudomonas aeruginosa , Proteus mirabilis , Klebsiella pneumoniae and Streptococcus faecalis from urinary-tract infections in spinally-injured patients together with Escherichia coli 10418 were challenged with chlorhexidine (200 mg l −1 ) in a model of a catheterized bladder under conditions which simulate the bladder washout technique. All species survived the antiseptic. Organisms growing on the wall of the bladder model appeared to be particularly resistant and electron microscopy showed that these cells were embedded in a protective glycocalyx. The effect of chlorhexidine bladder washouts on the bacterial flora in the urine of patients was also observed and shown to be minimal and temporary. Examination of urinary sediments from patients revealed the presence of micro-colonies of bacteria embedded in a polysaccharide matrix. We conclude that bladder washouts with chlorhexidine are not likely to eliminate established infections with organisms that occur in patients with indwelling bladder catheters.

In this study five laboratory media have been used to characterize the bacterial flora of urine from patients with spinal injuries who are undergoing long-term indwelling bladder catheterization. Single organisms were rarely isolated from these urines, in general complex mixed communities of bacteria were recovered composed of up to seven species. Observations were also made on how these populations change with time and in response to antibiotic therapy and bladder irrigation with antiseptics. During periods when patients were not receiving antibacterials, Escherichia coli , Klebsiella pneumoniae , Citrobacter diversus , Providencia stuartii , Pseudomonas aeruginosa , Proteus mirabilis and Pr. morganii were stable inhabitants of the urine, Streptococcus faecalis , Acinetobacter anitratus and Staphylococcus epidermidis were transient members of the communities. The effect of antibacterials on these communities was merely to select species that were resistant to the agents used. These results suggest that if these patients are to be treated, then efforts should be made to fully characterize the bacterial populations of the urines. Any therapy should then be based upon a consideration of the resistance profiles of all the members of the mixed bacterial flora.

The ability of urinary isolates of nine species of bacteria to bind to urinary catheters has been assessed using a bioluminescence technique. The experiments revealed the particularly adherent properties of Pseudomonas aeruginosa. This organism has also been shown to be most frequently isolated from the urines of the patients undergoing indwelling catheterization. It is suggested that P. aeruginosa commonly grows on the surface of catheters in situ, and that at this site it can survive antibiotic therapy and cause apparent recurrence of infection by reinoculation of the urine once therapy has been completed.