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A J Lastovica

Publications and source records attributed to A J Lastovica.

At least 19 recordsLinked to original sources
PubMed2006

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus.

AIM: DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. METHODS AND RESULTS: RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. CONCLUSIONS: Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.

Adult↗
PubMed2002

Genetic basis of quinolone resistance and epidemiology of resistant and susceptible isolates of porcine Campylobacter coli strains.

AIMS: The aims of this study were to investigate the epidemiology of quinolone-resistant and -susceptible porcine isolates of Campylobacter coli and to characterize the genetic basis of quinolone resistance. METHODS AND RESULTS: Penner serotyping and flagellin gene sequence polymorphisms were used to investigate the epidemiology of the C. coli isolates. A total of 55 isolates were included, of which 30 were paired resistant and susceptible isolates from 15 pigs. Amplification of gyrA, gyrB and parC, followed by direct sequencing of amplicons was used to identify mutations in the targets of quinolones. Overall, 31 of the isolates were resistant to ciprofloxacin (minimum inhibitory concentrations (MIC), 2- >or = 32 microg x ml(-1)). Thirteen DdeI-flaA profiles were observed and resistant and susceptible strains were identified for nine profiles. The majority of resistant strains exhibited either profile 1 or 6. While profile 1 comprised susceptible and resistant strains, all of the strains with profile 6 were resistant to ciprofloxacin. The serogroup (O:24) of the profile 6 strains was identical. The only other serogroup to be uniformly associated with quinolone resistance was O:5. Strains with this phenotype comprised a number of genotypes, including profile 1. Only four of the paired isolates from individual pigs had the same profile. The genetic basis of quinolone resistance was investigated in two strains with ciprofloxacin MICs of 2 and > or = 32 miccrog x ml(-1), respectively. The amino acid substitution of isoleucine for threonine at position 86 was identified in the GyrA proteins from both strains. No mutations were identified in the GyrB proteins. CONCLUSIONS: There was an association between two of the genotypes, serotypes 5 and 24, and quinolone resistance. The association between genotype, serotype and resistance in C. coli isolates has not been reported previously. Only the mutation in GyrA associated with quinolone resistance was identified. No mutations in GyrB were identified. Amplification products of parC were not obtained and it may be that this gene is not present in some Campylobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on the distribution of ciprofloxacin resistance between subtypes of C. coli.

Animals↗
PubMed2002

The pepsinogen releasing effect of Helicobacter pylori lipopolysaccharide.

BACKGROUND: Helicobacter pylori lipopolysaccharide (LPS) affects pepsinogen release by a nontoxic mechanism. We hypothesized that this effect was characteristic of the organism and related to the clinical status of the strain. MATERIALS AND METHODS: LPS was isolated from 11 H. pylori strains whose pathogenic profile was known and four other nongastric bacteria. The effects of luminal LPS on guinea pig gastric mucosal pepsinogen release was evaluated using the Ussing chamber technique. CCK-8 (10(-9)M) was used as a positive control. RESULTS: H. pylori LPS dose-dependently stimulated pepsinogen release with a maximal stimulation at 250 microg/ml (approximately 4500; p < .001 vs. control). LPS from other Helicobacter or Campylobacter species had no effect on pepsinogen release. ANOVA demonstrated significant differences in the efficacies of pepsinogen release between the 11 clinical H. pylori strains (p < .0001) despite the fact that they were all cagA+ and 90 had the cytotoxic vacA subtype s1. Physical and chemical disruption of the LPS suggested that both the structure and the carbohydrate composition of this molecule may play a critical role in pepsinogen release. Polymyxin B partly (p < .03) inhibited and dephosphorylation completely inhibited (p = .0002) LPS-stimulated pepsinogen release. CONCLUSION: Pepsinogen release is an innate property of all cagA+H. pylori LPS. The structure of the molecule and composition of side-chains are important in this response which appears to be partially lipid A driven.

Adult↗
PubMed2002

Evaluation of a novel heminested PCR assay based on the phosphoglucosamine mutase gene for detection of Helicobacter pylori in saliva and dental plaque.

A novel heminested PCR protocol was developed for the specific detection of Helicobacter pylori at low copy numbers. A set of primers specific for the phosphoglucosamine mutase gene (glmM) of H. pylori produced a 765-bp fragment that was used as template for the heminested primer pair delineating a 496-bp fragment. By using agarose gel electrophoresis for detection of the heminested PCR-amplified products, amplification of H. pylori genomic DNA was achieved at concentrations as low as 0.1 pg, equivalent to 5 x 10(2) bacteria. A study was subsequently undertaken to evaluate the heminested PCR for detection of H. pylori in dental plaque and saliva. Specimens collected from 58 individuals were cultured, and PCR was subsequently performed on the oral cultures. Identification of H. pylori in the same series of saliva and dental plaque specimens was carried out with PCR using a primer pair specific for the H. pylori urease B gene and by the heminested PCR assay. The identity of the amplified products was confirmed by DNA sequencing. Our results demonstrate that the heminested PCR assay was specific for detection of H. pylori, yielding no false-positive results, and that H. pylori had a low prevalence (approximately 3%) in specimens obtained from the oral cavity.

DNA Primers↗
PubMed2001

Molecular identification of Campylobacter concisus.

A 1.6-kb DNA fragment isolated from a Campylobacter concisus genomic library gave C. concisus-specific restriction fragment length patterns when it was used as a probe in hybridization studies. All of the strains tested, including type strains and clinical isolates, contained a 0.5-kb HindIII fragment that hybridized to the probe. DNA sequencing of the 1.6-kb fragment identified three open reading frames (ORFs). One of the ORFs encodes the carboxy terminus of GyrB, and the translational products of ORF2 and ORF3 showed similarity to hypothetical proteins, previously identified in Campylobacter jejuni. DNA-DNA hybridization studies with a fragment internal to ORF3 showed that this sequence was responsible for the signal observed with the 0.5-kb HindIII fragment. A rapid PCR assay was developed and evaluated. Primers that annealed to the extremities of the 1.6-kb fragment were used to obtain an amplicon of the correct size from both reference and clinical strains of C. concisus.

Adult↗
PubMed2001

Clustering of South African Helicobacter pylori isolates from peptic ulcer disease patients is demonstrated by repetitive extragenic palindromic-PCR fingerprinting.

The present report assesses the association between clonal groupings, disease, and the virulence fingerprint of 76 South African Helicobacter pylori cagA(+) strains isolated from 57 Cape-colored subjects. Two methods, repetitive extragenic palindromic (REP)-PCR and random amplified polymorphic DNA (RAPD)-PCR, were used to generate DNA fingerprints, and computer-assisted analysis was used to derive clusters. The two PCR techniques were only partially complementary (48%). REP-PCR fingerprints identified a distinct pathological cluster consisting of strains from 63% of the patients and was strongly associated with both disease (P < 0.00001) and the vacuolating cytotoxin A (vacA) signal sequence type (P < 0.003). RAPD-PCR fingerprinting was not associated with disease and was less strongly associated with vacA (P < 0.05) than REP-PCR was. Hierarchical analysis indicated that isolates from patients with peptic ulcer disease tended to cluster differently than isolates from patients with gastritis alone or gastric adenocarcinoma. These relationships are consistent with a loosely clonal population structure associated with disease for H. pylori in the Cape-colored population in South Africa.

Adolescent↗
PubMed2001

Conservation of the cag pathogenicity island is associated with vacA alleles and gastroduodenal disease in South African Helicobacter pylori isolates.

BACKGROUND: The development of clinically significant disease in South Africa is associated with the vacuolating cytotoxin gene (vacA) s1 genotype but not with the presence of the cytotoxin associated gene cagA. cagA occurs in >95% of South African isolates and is a variable marker for the entire cag pathogenicity island (PAI). AIM: To characterise the cagPAI in South African isolates and to investigate if structural variants of this multigene locus were associated with variations in vacA status and clinical outcome. PATIENTS AND METHODS: We studied 109 Helicobacter pylori strains (36 from patients with peptic ulceration, 26 with gastric adenocarcinoma, and 47 with no pathology other than gastritis) for differences in selected genes of the cagPAI and alleles of vacA by polymerase chain reaction. RESULTS: All strains were cagA(+). Sixty five (60%) strains had an intact contiguous cagPAI; 78% of peptic ulcer isolates, 73% of gastric adenocarcinoma isolates, but only 40% of gastritis alone isolates (p< 0.01). The entire cagII region was undetectable in 23% of gastritis alone isolates but in only 8% of peptic ulceration isolates (p<0.05). The vacA signal sequence and mid region demonstrated a strong relationship between the virulence associated vacA s1 (p<0.005) and vacA m1 (p=0.05) alleles and an intact cagPAI. CONCLUSION: Although a complete cagPAI was a feature of most infected individuals, deletions in the 5' region of this genetic locus were associated with gastritis alone and with the non-cytotoxic s2/m2 vacA genotype.

Adenocarcinoma↗
PubMed2001

Analysis of iceA genotypes in South African Helicobacter pylori strains and relationship to clinically significant disease.

BACKGROUND: South African Helicobacter pylori isolates are characterised by the universal presence of cagA but have differences in vacuolating cytotoxin gene (vacA) alleles which correlate with clinically significant disease. However, the candidate virulence marker gene iceA has not been investigated. AIM: To characterise the genetic organisation and heterogeneity of iceA genotypes in different South African clinical isolates. PATIENTS AND METHODS: We studied H pylori strains isolated from 86 dyspeptic patients (30 with peptic ulcer disease (PUD), 19 with distal gastric adenocarcinoma (GC), and 37 with non-erosive gastritis) for the presence of iceA1 or iceA2 genes, and for differences in the genetic organisation of iceA2 by polymerase chain reaction, Southern hybridisation analysis, and sequencing. RESULTS: Genetic analysis of iceA1 demonstrated significant homology (92-95%) with the USA type strain 26695 and probably functions as a transcriptional regulator, while a novel variant (iceA2D') of iceA2 and marked differences in predicted protein secondary structure of the iceA2 protein were defined. iceA1 was detected in 68% and iceA2 in 80% of all clinical isolates. Although approximately 40% of patients had both strains, a higher prevalence (p< 0.01) of GC patients were infected with iceA1 isolates which were invariably vacA s1/iceA1 (p< 0.005 v gastritis). Isolates from PUD patients were distinguished by the structurally altered iceA2D variant (53%; p<0.03 v gastritis) while the iceA2C variant distinguished isolates from patients with gastritis alone (67%; p< 0.005 v PUD). CONCLUSION: In this study, an association between iceA1 and GC was noted while differences in variants of iceA2 differentiated between PUD and gastritis alone. Combination analyses of iceA genotypes and vacA alleles supported these associations.

Adenocarcinoma↗
PubMed2000

Genetic and biochemical evidence of a Campylobacter jejuni capsular polysaccharide that accounts for Penner serotype specificity.

Campylobacter jejuni, a Gram-negative spiral bacterium, is the most common bacterial cause of acute human gastroenteritis and is increasingly recognized for its association with the serious post-infection neurological complications of the Miller-Fisher and Guillain-Barré syndromes. C. jejuni lipopolysaccharide (LPS) is thought to be involved in the pathogenesis of both uncomplicated infection and more serious sequelae, yet the LPS remains poorly characterized. Current studies on C. jejuni suggest that all strains produce lipooligosaccharide (LOS), with about one-third of strains also producing high-molecular-weight LPS (referred to as O-antigen). In this report, we demonstrate the presence of the high-molecular-weight LPS in all C. jejuni strains tested. Furthermore, we show that this LPS is biochemically and genetically unrelated to LOS and is similar to group II and group III capsular polysaccharides. All tested kpsM, kpsS and kpsC mutants of C. jejuni lost the ability to produce O-antigen. Moreover, this correlated with serotype changes. We demonstrate for the first time that the previously described O-antigen of C. jejuni is a capsular polysaccharide and a common component of the thermostable antigen used for serotyping of C. jejuni.

ATP-Binding Cassette Transporters↗
PubMed2000

Genetic characterization of Campylobacter jejuni O:41 isolates in relation with Guillain-Barré syndrome.

Campylobacter jejuni O:41 strains are found in association with Guillain-Barré syndrome in South Africa. Strains of this serotype collected over 17 years were characterized by amplified fragment length polymorphism and flagellin typing to determine their clonal nature. Despite minor variation in GM1 expression, all of the strains were genetically indistinguishable, indicating that they are representative of a genetically stable clone.

Campylobacter Infections↗
PubMed1999

Heterogeneity in the Helicobacter pylori vacA and cagA genes: association with gastroduodenal disease in South Africa?

BACKGROUND: Helicobacter pylori infection is universally associated with gastritis, but only sometimes with clinically significant disease. Candidate virulence markers seem to be useful in identifying the pathogenic infections in some populations. AIMS: To investigate the association between putative virulence markers and disease in an African population. METHODS: Fifty nine H pylori strains isolated from dyspeptic patients (11 with peptic ulceration, eight with gastric adenocarcinoma, and 28 with no pathology other than gastritis) were studied for differences in the genes vacA and cagA. RESULTS: Forty seven (80%) of 59 strains had the vacA signal sequence genotype s1 (one s1a, 46 s1b) and 12 (20%) had subtype s2. vacA mid-region analysis revealed that 40 (68%) strains were vacA m1 and 19 (32%) were m2. All 14 strains from patients with peptic ulceration were vacA s1, in contrast to 23 (66%) of 35 strains from patients with gastritis alone (p<0.01). vacA s2 was found exclusively in patients with gastritis alone (p<0.01). All strains isolated from patients with gastric adenocarcinoma were s1b/m1 (p<0. 005 versus gastritis alone). cagA was detectable in 56 (95%) of 59 isolates. Strains from patients with peptic ulceration (12/13 versus 19/30 with gastritis alone, p=0.05) had the shortest fragment length in the 3' region of cagA, while 4/10 strains from patients with gastric cancer had the longest fragment length in this region (p<0. 02 versus gastritis alone). CONCLUSION: In this study, the vacA s1 genotype, and fragment length of the 3' region of cagA identified isolates associated with significant clinical disease. The vacA s1bm1 genotype seems to be strongly associated with gastric cancer.

Adenocarcinoma↗
PubMed1998

Lipopolysaccharides from Campylobacter jejuni O:41 strains associated with Guillain-Barré syndrome exhibit mimicry of GM1 ganglioside.

Three Campylobacter jejuni, biotype 2, serotype O:41 strains that were isolated from patients who developed Guillain-Barré syndrome (GBS) and one C. jejuni isolate from a patient who developed enteritis only were examined. The aim of the study was to determine the structure of the core oligosaccharide (OS) of the lipopolysaccharide (LPS) of C. jejuni serotype O:41, a serotype rarely associated with the development of GBS, and to determine if the LPS shares similar epitopes with any of the major human gangliosides. Electrophoretic analysis with silver staining or immunoblotting demonstrated that the strains had LPS profiles characteristic of low-molecular-weight LPS. Colorimetric analysis detected N-acetylneuraminic (sialic) acid in the core OSs of all the strains. Thin-layer chromatography with immunostaining showed that antisera raised against the GBS strains reacted with the GM1 ganglioside, suggesting that C. jejuni serotype O:41 LPSs and the GM1 ganglioside have similar epitopes. Furthermore, polyclonal anti-GM1 and anti-asialoGM1 antibodies cross-reacted with each C. jejuni O:41 LPS tested, suggesting that the serotype O:41 core OS has a GM1- and asialoGM1-like structure. LPSs extracted from C. jejuni serostrains O:2, O:3, and O:19 were also used in the study. Cholera toxin (a GM1 ligand) and peanut agglutinin (a Galbeta1-3GalNAc ligand) recognized all serotype O:41 LPSs and the serostrain O:2 LPS. Immunoadsorption results confirmed GM1 relatedness. Moreover, the core OS was isolated from a GBS-associated C. jejuni O:41 LPS by gel permeation chromatography. An analysis by gas-liquid chromatography (GLC), GLC-mass spectrometry, and nuclear magnetic resonance showed the core OS of one of the C. jejuni O:41 GBS isolates to have a tetrasaccharide structure consistent with GM1 mimicry.

Adult↗
PubMed1998

Microtiter assay for detecting Campylobacter spp. and Helicobacter pylori with surface gangliosides which bind cholera toxin.

Campylobacter jejuni with Gm1 ganglioside in the core of its lipopolysaccharide has been associated with Guillain-Barré syndrome. Since this epitope may be of considerable pathophysiologic importance and since this ganglioside binds cholera toxin, a rapid screening assay to detect bacteria that bind cholera toxin as an indication of Gm1 on their surfaces was developed. In the assay, bacterial lawns were grown on agar plates, harvested with phosphate-buffered saline, boiled, and incubated with a standard concentration of cholera B subunit. Preparations from strains with Gm1 were observed to inhibit the binding of cholera B subunit to Gm1 in a microtiter enzyme-linked immunosorbent assay. By using this assay with two groups of strains, 37 positive strains were detected among the 197 tested. Species with positive isolates included C. jejuni, Campylobacter coli, and Helicobacter pylori. The assay is capable of testing large numbers of isolates and should prove useful in future clinical and epidemiological studies of bacteria with this epitope.

Bacteriological Techniques↗
PubMed1997

Guillain-Barré syndrome in South Africa associated with Campylobacter jejuni O:41 strains.

Over a 20-month period, 3 adult and 6 pediatric patients were diagnosed with Guillain-Barré syndrome (GBS) at Groote Schuur and Red Cross Hospitals in Cape Town. All 9 GBS patients had Campylobacter jejuni biotype 2, serotype O:41 in their stools. C. jejuni infection was confirmed by ELISA testing of patient sera. Strains of this sero-biotype are rare: Only 12 such strains, including the GBS-associated strains, were recognized among 776 Campylobacter strains isolated and identified at Red Cross Hospital from March 1994 to October 1995. This is the first known association of C. jejuni biotype 2, serotype O:41 with GBS. Patients infected with this Campylobacter strain had a particularly severe form of the infection, requiring hospitalization and ventilation much longer than GBS patients infected with other Campylobacter species and patients with Campylobacter-negative stools. The O:41 Campylobacter isolates from the GBS patients are identical by phenotypic, serologic, and molecular criteria, and they are clonal.

Adolescent↗