The status of antimicrobial resistance of Helicobacter pylori in eastern Europe.

OBJECTIVE: To evaluate the primary, secondary and combined resistance to five antimicrobial agents of 2340 Helicobacter pylori isolates from 19 centers in 10 countries in eastern Europe. METHODS: Data were available for centers in Bulgaria, Croatia, the Czech Republic, Estonia, Greece, Lithuania, Poland, Russia, Slovenia and Turkey. Susceptibility was tested by agar dilution (seven countries), E test (five countries) and disk diffusion (three countries) methods. Resistance breakpoints (mg/L) were: metronidazole 8, clarithromycin 1, amoxicillin 0.5, tetracycline 4, and ciprofloxacin 1 or 4 in most centers. Primary and post-treatment resistance was assessed in 2003 and 337 isolates respectively. Results for 282 children and 201 adults were compared. RESULTS: Primary resistance rates since 1998 were: metronidazole 37.9%, clarithromycin 9.5%, amoxicillin 0.9%, tetracycline 1.9%, ciprofloxacin 3.9%, and both metronidazole and clarithromycin 6.1%. Isolates from centers in Slovenia and Lithuania exhibited low resistance rates. Since 1998, amoxicillin resistance has been detected in the southeastern region. From 1996, metronidazole resistance increased significantly from 30.5% to 36.4%, while clarithromycin resistance increased slightly from 8.9% to 10.6%. In centers in Greece, Poland, and Bulgaria, the mean metronidazole resistance was slightly higher in adults than in children (39% versus 31.2%, P > 0.05); this trend was not found for clarithromycin or amoxicillin (P > 0.20). Post-treatment resistance rates exhibited wide variations. CONCLUSIONS: In eastern Europe, primary H. pylori resistance to metronidazole is considerable, and that to clarithromycin is similar to or slightly higher than that in western Europe. Resistance to amoxicillin, ciprofloxacin and tetracycline was detected in several centers. Primary and post-treatment resistance rates vary greatly between centers.

Primary resistance to clarithromycin in clinical strains of Helicobacter pylori isolated from children in Poland.

Helicobacter pylori resistance to clarithromycin is an important factor in the failure of eradication therapy. The resistance results from point mutations in the 23S rRNA gene of H. pylori. The prevalence of primary resistance of H. pylori to clarithromycin in children and mutations associated with resistance were studied and it was found that 23.5% (23/98) of H. pylori strains isolated in our hospital during 1998-2000 were resistant to clarithromycin. The primary resistance was mainly caused by an A2143G mutation, but the isolates with an A2142G mutation had higher MICs for clarithromycin compared with those with an A2143G mutation: median MIC 256 versus 16 mg/l. Comparison of our data with previous results showed that the prevalence of H. pylori resistance to clarithromycin in children has increased in Poland over the last three years, however the difference was not significant (23.5 vs. 17%, P=0.22).

[Antagonistic activity of Lactobacillus bacteria strains against anaerobic gastrointestinal tract pathogens (Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Clostridium difficile)]. / Antagonistyczne dzialanie bakterii z rodzaju Lactobacillus wobec beztlenowych i mikroaerofilnych czynników zakazen przewodu pokarmowego (Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Clostridium difficile).

Antagonistic activity of Lactobacillus strains has been known for some time. This property is connected with production of many active substances by lactobacilli e.g., organic acids and bacteriocin-like substances which interfere with other indigenous microorganisms inhabiting the same ecological niche, including also anaerobic gastrointestinal tract pathogens. Growing interest of clinical medicine in finding new approaches to treatment and prevention of common inflammatory infections of the digestive tract resulted in studies on a possible usage of lactic acid bacteria. Last years, several in vitro and in vivo experiments on antagonism of different Lactobacillus strains against Helicobacter pylori and Clostridium difficile were performed. These observations had been done on already established, well known probiotic Lactobacillus strains. We tested antibacterial activities of Lactobacillus strains isolated from human digestive tract. As indicator bacteria, four species known as anaerobic bacterial etiologic agents of gastroenteric infections: Helicobacter pylori, Campylobacter jejuni, C. coli and Clostridium difficile were used. Some of them were obtained from international collections, others were clinical isolates from specimens taken from patients with different defined gastrointestinal infections. We used a slab method of testing inhibitory activity described in details previously. Following conclusions were drawn from our study: All tested human Lactobacillus strains were able to inhibit the growth of all strains of anaerobic human gastrointestinal pathogens used in this study. Inhibitory activities of tested Lactobacillus strains against Helicobacter pylori, Campylobacter spp., and Clostridium difficile as measured by comparing mean diameters of the inhibition zones were similar. Differences in susceptibility of individual indicator strains of Campylobacter spp. and Clostridium difficile to inhibitory activity of Lactobacillus strains were small. A similar mechanism of inhibition of anaerobic bacteria by lactobacilli is postulated.

Genetic diversity of the Campylobacter genes coding immunodominant proteins.

Three Campylobacter jejuni 72Dz/92 genes (cjaA (ompH1), cjaC (hisJ) and cjaD (omp18)) encoding immunodominant proteins are considered to be potential chicken vaccine candidates. The presence and conservation of cjaA, cjaC and cjaD genes among different Campylobacter clinical isolates were determined. The genes were detected in thirty Campylobacter strains using hybridization as well as Western blot analysis. However, PCR products of the predicted size were amplified only from ten out of thirty examined strains regardless of the employed primer pair. The nucleotide sequence of the C. jejuni 72Dz/92 genes was compared with the nucleotide sequences of their homologs cloned from other Campylobacter strains as well as with the whole genome sequence of C. jejuni NCTC 11168. The examined sequences revealed 0 to 16% divergence. Strain-dependent levels of divergence were observed. The polymorphism detected in cjaC was mainly within the 5' region of the gene, while the nucleotide substitutions in cjaA and cjaD are distributed uniformly along the whole genes. Most of the observed nucleotide substitutions occurred at the third base of the codons. This observation is consistent with the results of Western blot experiments.

PCR-based diagnosis of Helicobacter pylori infection in Polish children and adults.

Infection and associated disease caused by Helicobacter pylori are common in Poland, as in much of Eastern Europe, although the genotypes of strains have not been much studied, especially in terms of traits that might be important in disease. This study developed a sensitive and efficient polymerase chain reaction (PCR) test for the presence of H. pylori in gastric biopsy samples with ureA gene-specific primers and primers for the virulence-associated cag pathogenicity island (PAI). These tests were used with biopsy samples from 246 symptomatic children (age range 1-17 years) and 82 adults (age range 18-53 years) in Warsaw. An assessment was also made of the success of metronidazole-based therapy intended to eradicate infection. H. pylori was detected by ureA-specific PCR in 83 (76.9%) children and in 41 (87.2%) adults with histologically proven gastritis, and in 28.4% and 29.2%, respectively, of the 38 children and 7 adults with little or no evidence of gastritis. In general, H. pylori was detected more often by PCR than by culture (70.3% compared with 52.8% in children and 62.8% compared with 38.6% in adults), although in several cases a negative PCR was associated with a positive culture result. The rate of H. pylori infection increased with age from 5.4% in children up to 5 years old to 29.2% to age 10 and 65.4% to age 18. The tests detected the cagPAI in 97 (75%) and 44 (85%) of the H. pylori-infected children and adults, respectively. Some H. pylori-infected patients with a ureA+ PCR result contained the 'empty site' of the cagPAI and only four patients were infected with mixed cag+ cag- strains. PCR with cagPAI and 'empty site' of the cagPAI represents a novel tool for fast screening of mixed cag+ cag- infection. These results confirm and further illustrate that direct PCR of biopsy specimens can be useful for detection of infection and genotyping of resident strains, and that H. pylori infection is very common among children as well as adults in Poland. They also show that Polish strains vary with regard to the presence or absence of the cagPAI, and suggest that the proportion of strains that are cag+ is higher in Poland than in Western European countries, which may reflect the relatively higher risk of infection in this society.

Genotypes of Helicobacter pylori in Polish population.

Here we have studied the genetic diversity of Helicobacter pylori strains recovered from 64 individual patients, 5 family members and 13 unsuccessfully treated patients. The recovered bacteria were finger-printed by the PCR-RFLP and RAPD methods and virulence associated loci (cagPAI, vacA) were PCR studied. Unique differentiation of every independently isolated strain from not-related persons was possible by RAPD technique. In PCR-RFLP technique several profile groups (7 and 15) for particular endonuclease tested were found. Eleven patients carried strains of the same gene profile (PCR-RFLP) and the same overall genotype (RAPD) before and after therapy. In the family studies, essentially the same strain was found in different relatives in three cases, and different strains were found in the other two cases. Island of cagPAI was present in 79% of all strains tested, half and one-fifth of all strains tested presented, s1am2 and s1m1 alleles of vacA gene, respectively. Independently from identity or diversity of pre- and post-treatment strains and strains recovered from the family members we have been observed identical cagPAI/vacA genotypes. These results suggest that H. pylori infections in Poland can be mixed, although just one strain may often predominate, and that inter-family transmission may be significant even in this high risk society. The genetic feature of virulence-associated loci are similar to those seen elsewhere in Europe, although strains that carry the cagPAI and the potentially more toxigenic alleles of the vacA gene are more common. RAPD technique is proven as most differentiating, however PCR-RFLP allows for easy recognition of mixed infection with two or more different strains. Molecular typing study in case of children therapy may allow reduce rate of relapses by reduction of possible transmission from family source.

Cloning and characterization of a Campylobacter jejuni 72Dz/92 gene encoding a 30 kDa immunopositive protein, component of the ABC transport system; expression of the gene in avirulent Salmonella typhimurium.

Three gene libraries of Campylobacter jejuni 72Dz/92 DNA were prepared using lambda gt11, pSupercos and pWSK129 cloning vectors. Screening of the libraries with Escherichia coli absorbed antiserum generated against whole C. jejuni revealed several immunoreactive clones of apparent molecular masses 19, 28, 30 and 50 kDa. The most commonly isolated clones expressed 30 kDa protein. The nucleotide sequence of the 1768 bp C. jejuni DNA yielded one complete (ORF2) and two partial open reading frames (ORF1 and ORF3). ORF2 encoded CjaA protein exhibits relevant overall homology to several prokaryotic solute binding proteins (family 3), components of the ABC transport system, while the product of the truncated ORF3 (CjaB protein) shows extensive homology to Gram-negative bacterial proteins, members of the sugar transporter family. The genetic organization of the putative cjaAB operon was studied. The cjaA gene fragment (616 bp) was amplified from three C. jejuni strains isolated from patients with acute bloody diarrhea, whereas it was not amplified from strains which caused acute diarrhea with no blood in the stools. The gene was introduced into avirulent Salmonella typhimurium vaccine strain where it is expressed at a reasonably high level.

PCR for identification and typing of Helicobacter pylori isolated from children.

From 191 children with upper gastroduodenal disease 236 gastric biopsy specimens were taken. H. pylori was detected by use of culture Gram staining histological examination and PCR technique. A segment of DNA coding protein synthesis of 26 kDa or urease A and B gene were used for PCR amplification. PCR technique was also used for determination of the presence of Cag A gene in 72 strains of H. pylori isolated from children. Genetic typing of H. pylori strains by RFLP analysis of PCR amplified urease B gene 933 bp fragment and RAPD were performed. Biopsy specimens taken from children with gastritis were in 52% H. pylori culture and PCR positive, while 18.1% PCR positive only. Similarly, specimens taken from children with duodenal ulcer were in 50% H. pylori culture and PCR positive, while 12.5% PCR positive only. Fifty one (70.8%) from 72 strains of H. pylori were Cag A positive. Molecular typing of the strains isolated during first and follow-up endoscopy allowed the differentiation between reinfection or new infection and coinfection. It was shown that RAPD typing had better discrimination power in comparison to PCR--RFLP method.

Antibiotic resistance of Campylobacter jejuni with reference to plasmid profiles of clinical and chicken isolates.

A total of 47 clinical isolates and 52 poultry isolates of Campylobacter jejuni were characterized by their resistance to 16 antimicrobial agents and by plasmid profiles on agarose gel electrophoresis. Almost all isolates were susceptible to erythromycin, chloramphenicol, gentamycin and nitrofuratoin. Plasmids were detected in 19% of C. jejuni strains isolated from feces of children patients and in 36% of strains isolated from chicken. The presence of plasmid DNA was not found to be correlated with any definite resistance, despite of the number of resistant strains was also higher among poultry isolates. Plasmids, as well, does not seem to be essential for colonization of alimentary tract of pathogenic activity.

Characterization of Campylobacter jejuni asd gene cloned in Escherichia coli.

asd gene of the human enteric pathogen, Campylobacter jejuni Dz72/92, has been isolated from a genomic library constructed in the expression plasmid vector pUC8-2 in Escherichia coli. The gene has been fished out by complementation of the asd gene deletion present in the genome of E. coli chi 6097. The smallest recombinant plasmid (pUWM3) able to confer Asd+ phenotype contains a 1.8 kb insert cloned into HindIII site located within the multi-cloning site of the pUC8-2 vector. The origin of the insert has been confirmed by hybridization. Several pieces of evidence indicate that the expression of the cloned house-keeping gene is driven from its own promoter, which can be recognised by E. coli RNA polymerase. The asd gene promoter has been located on 300 kb HindIII-DraI fragment of pUWM3. Recombinant plasmid pUWM3 specifies a new 38 kDa protein which we believe is the asd gene product. Overproduction of the 38 kDa protein due to the transcription originating from the vector lacZ gene promoter is toxic for the cells.

[Biochemical and serologic characteristics of Campylobacter jejuni/coli strains causing diarrhea in children]. / Biochemiczna i serologiczna charakterystyka szczepów Campylobacter jejuni/coli odpowiedzialnych za biegunki u dzieci.

One hundred strains of Campylobacter jejuni/coli isolated from faeces of children with diarrhoea were characterised. Frequency of isolation of these microorganisms from faeces of children with enterocolitis symptoms was evaluated. In this group Campylobacter jejuni/coli constituted 11.4% of all isolates, being the dominant etiologic agent of these infections. Biotype pattern of 100 Campylobacter jejuni/coli strains was determined. Biotype I C.jejuni prevailed and C. coli constituted as much as 35% of all isolated strains. All isolated strains were characterised serologically according to typing scheme of Lior. Seventy four strains were typed and 22 were untypable, out of which four were rough. Two new serotypes were isolated: LIO 71 and LIO 72, LIO 4 and LIO 72 serotypes were the most frequently isolated. Frequency of isolation of Campylobacter jejuni/coli strains were also determined in the period from january 1985 to august 1987.