Mixed-infection of antibiotic susceptible and resistant Helicobacter pylori isolates in a single patient and underestimation of antimicrobial susceptibility testing

Antibiotic resistance among Helicobacter pylori has been increasing worldwide and has begun to affect the overall efficacy of current antibiotic regimens adversely. We examined 220 pairs of H. pylori isolates obtained from both the antrum and corpus of separate patients; 109 (50%) harbored antibiotic-resistant H. pylori: amoxicillin (0.5%), clarithromycin (5.9%), furazolidone (1.4%), metronidazole (45.5%), nitrofurantoin (1.4%), and tetracycline (6.8%). Heteroresistance among the two biopsy sites from each patient was present in 41 of the 109 patients (38%) with antibiotic resistant H. pylori (e.g. 34% with resistant strains would be misclassified as susceptible if a biopsy of the antrum alone used for antimicrobial susceptibility testing). DNA fingerprinting genotype analysis was carried out on the 41 pairs of isolates with heteroresistance. While different patients had different fingerprinting patterns, each pair of isolates showed identical or similar fingerprinting patterns. These results suggest that antibiotic-resistant H. pylori typically develop from pre-existing susceptible strain rather than coinfection with a different strain. The minor differences in genotype (degeneration of genotype) seen reflect one of the processes for development of genetic diversity in H. pylori. No biopsy single site can be considered representative for antimicrobial susceptibility testing.     

Effectiveness of enterobacterial repetitive intergenic consensus PCR and random amplified polymorphic DNA fingerprinting for Helicobacter pylori strain differentiation

We compared the robustness and discriminatory power of the enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphic DNA (RAPD) fingerprinting methods for detecting cases of mixed Helicobacter pylori infection in Peruvian shantytown residents. H. pylori isolates from 63 participants were cultured, and five single colonies and a pool of additional colonies from each participant were analyzed by ERIC-PCR and by RAPD tests with four 10-nucleotide primers (one primer per reaction). There was 94% agreement between the ERIC and RAPD profiles in classifying sets of isolates as uniform versus closely related but not identical versus probably unrelated, indicating a high kappa statistic of 0.8942. Subtle differences in related ERIC or RAPD patterns likely reflect gene transfer between strains, recombination, and/or mutation, whereas markedly different patterns reflect infection by unrelated strains. At least half of infected shantytown residents seemed to carry more than one H. pylori strain, although in 19 of 31 persons, the strains were closely related. Three RAPD tests, each with a different primer, were needed to achieve the sensitivity of one ERIC test. ERIC-PCR constitutes a resource- and time-efficient method for H. pylori strain differentiation.     

Molecular fingerprinting of Helicobacter pylori strains from duodenal ulcer patients

AIMS: To characterize the molecular fingerprinting of Helicobacter pylori population isolated in duodenal ulcer patients treated with triple therapy. METHODS AND RESULTS: Gastric biopsy specimens from corpus and antrum, were cultured for H. pylori isolation. Helicobacter pylori eradication was evaluated after 4 and 16 weeks. DNAs of all isolates were characterized by random amplified polymorphic DNA typing and cagA gene was also detected. After the therapy, five patients harboured the microorganism at 4 weeks and two of them remained H. pylori positive at 16 weeks. The analysis of DNA fingerprinting of strains isolated from antrum and corpus of patients susceptible to treatment, showed similar patterns. Instead, when the therapy was not effective, strains isolated from sequential biopsies from initial and after 4 and 16 weeks, showed distinct fingerprintings and retained the cagA status, over time. CONCLUSIONS: The drugs used for therapy could exercise an effect in genotypical rearrangement among H. pylori cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The variableness among H. pylori strains represents a way to challenge environmental stress.     

幽门螺杆菌临床分离株的随机扩增多态性DNA指纹图分析

目的:建立武汉及周边地区幽门螺杆菌(Helicobacter pylori,Hp)感染病人胃内分离的Hp的DNA指纹图谱,并进行数理统计分析,探讨Hp基因型与疾病的相互关系,为临床诊断、防治及致病机制提供理论与实践基础.方法:采取随机扩增多态性DNA指纹法(Random amplified polymorphic DNA,RAPD)对48例病人Hp基因组DNA进行PCR反应,其随机引物为:1290 5'-GTGGATGCGA-3'.反应产物经2%琼脂糖凝胶电泳,成像存盘.用统计分析软件(Statistic analysis software,SAS)对Hp DNA指纹图的相似性以及与疾病的相关性进行分析.结果:每个菌株都有其独特的DNA指纹图,显示其基因的多态性;计算机聚类分析显示:Hp DNA指纹图可分为两大类,其与宿主疾病之间有一定程度的相关性(P＜0.05).结论:(1)RAPD对 Hp DNA扩增结果是稳定、可靠的,是一种较好的分型方法.(2)幽门螺杆菌感染所致疾病可能与其基因型相关.     

The rdxA gene plays a more major role than frxA gene mutation in high-level metronidazole resistance of Helicobacter pylori in Taiwan

BACKGROUND: Metronidazole-resistant H. pylori associating with mutations of rdxA or frxA is still a debated topic. This study investigates whether rdxA and frxA mutations of H. pylori accounted for the high MIC value (>/= 64 micro g/ml) of metronidazole (Mtz). MATERIAL AND METHODS: From 126 clinical H. pylori isolates, we examined 14 Mtz-sensitive, 18 Mtz-resistant H. pylori, and eight pairs of Mtz-sensitive and Mtz-resistant colonies simultaneously present within a single gastric biopsy. The paired strains from one single biopsy were proven identical by PCR-RFLP. MICs of Mtz were checked by the E-test and agar dilution method. The mutations of rdxA and frxA sequencing were matched with the Mtz-susceptible ATCC 26695 and J99. RESULTS: There were 89% (16/18) of Mtz-resistant isolates with mutation of RdxA. Half of the 14 Mtz-sensitive strains, all without mutation of RdxA, still contained truncation of FrxA. Within the paired isolates from a single biopsy, rdxA mutation (86%) was more common than frxA mutation (43%) in those isolates with high-level Mtz-resistant H. pylori. RdxA truncation was more prevalent in Mtz-resistant strains with high MICs than in those with low to moderate MICs (75% vs. 20%, p =.01, OR: 12, 95% CI: 1.8-81.7). CONCLUSION: Mutations in the rdxA gene rather than the frxA gene generally determine a high MIC level of Mtz-resistant H. pylori in Taiwan.     

DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting.

The RAPD (or AP-PCR) DNA fingerprinting method was used to distinguish among clinical isolates of Helicobacter pylori, a bacterium whose long term carriage is associated with gastritis, peptic ulcers and gastric carcinomas. This method uses arbitrarily chosen oligonucleotides to prime DNA synthesis from genomic sites to which they are fortuitously matched, or almost matched. Most 10-nt primers with > or = 60% G + C yielded strain-specific arrays of up to 15 prominent fragments, as did most longer (> or = 17-nt) primers, whereas most 10-nt primers with 50% G+C did not. Each of 64 independent H. pylori isolates, 60 of which were from patients in the same hospital, was distinguishable with a single RAPD primer, which suggests a high level of DNA sequence diversity within this species. In contrast, isolates from initial and followup biopsies were indistinguishable in each of three cases tested.     

Clarithromycin resistance in Helicobacter pylori strains isolated from French children: prevalence of the different mutations and coexistence of clones harboring two different mutations in the same biopsy

BACKGROUND: The aim of our study was to assess the different mutations involved in clarithromycin-resistant Helicobacter pylori strains isolated from French children and their temporal trends. METHODS: The point mutations of H. pylori were detected by PCR followed by RFLP technique in 50 clarithromycin-resistant strains collected between 1993 and 2004 in France. RESULTS: Clarithromycin resistance was observed in 23% (50/217) of H. pylori isolates. Two mutations A2143G and A2142G in the 23S rRNA genes of H. pylori were detected. The former was found in 45/50 (90%) of isolates. The rate of resistance increased with time from 18.6% in the period 1993-1996 to 41.6% in 2001-2004. No significant difference was observed in the distribution of mutations during the same periods. No correlation was found between any mutation and age, sex, and ethnic origin of children. Furthermore, no significant differences in minimal inhibitory concentrations level were observed according to the different point mutations. In all cases, only one point mutation was present, except in two cases where two different mutations were found in two different clones from the same biopsy. CONCLUSION: The mutation A2143G is predominant in clarithromycin-resistant H. pylori strains isolated from children in France. We report for the first time the presence of two clarithromycin-resistant clones harboring two different mutations of the 23S rRNA genes present in the same biopsy specimen and genotypically identical as demonstrated by RAPD fingerprinting.     

Intrafamilial spread of Helicobacter pylori: a genetic analysis

Background. A high incidence of Helicobacter pylori among family members of children with H. pylori gastritis has previously been documented on biopsy material. The main objective of this study was the genetic clarification of H. pylori strains involved in intrafamilial dispersion. Materials and Methods. Formalin‐fixed, paraffin‐embedded material of antral mucosa from 32 members of 11 families was studied for the presence of genetic homogeneity. To achieve this goal, the entire genome of H. pylori was studied by the polymerase chain reaction (PCR)‐based random amplified polymorphic DNA (RAPD) fingerprinting method. Furthermore, the Urease A gene was analyzed using a multiplex PCR‐assay and an alternative mutation detection method based on the Hydrolink? analysis. Results. RAPD fingerprinting confirmed that closely related H. pylori strains were involved in the intrafamilial dispersion. Mutations and small deletions in Urease A gene were found in 22 out of 32 individuals. Conclusions. The homology of the H. pylori genome in members of the same family strongly supports the hypothesis of transmission of H. pylori from person‐to‐person or from a common source.     

Typing of methicillin-resistant Staphylococcus aureus (MRSA) strains from an intensive care unit by random amplified polymorphic DNA (RAPD).

The identification and control of methicillin-resistant Staphylococcus aureus (MRSA) is of primary concern in intensive care units (ICUs) worldwide. The introduction and circulation of particular strains is best studied by genomic procedures and random amplified polymorphic DNA (RAPD) is well suited for this task. In this study 14 isolates of MRSA, obtained over an 8 month period from the blood cultures of 12 patients in an ICU at our hospital, were typed by RAPD method using seven primers. Three separate groups were distinguished and clustering of certain types in time and space was noted. These results suggest that although different strains of MRSA were involved in this outbreak, cross-infection with individual types occurred. RAPD fingerprinting is a relatively simple method that allows epidemiologic investigation of MRSA outbreaks in hospital infection.     

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.     

Superoxide dismutase activity of Helicobacter pylori per se from 158 clinical isolates and the characteristics

We investigated the correlation between the SOD activity of Helicobacter pylori (H. pylori) and gastroduodenal diseases and the characteristics of strains exposed to oxidative stress. Two sequenced strains, 26695 and J99, and clinical isolates from 156 Japanese patients with gastroduodenal diseases such as gastric cancer (n= 59) and non-cancer (n= 97) were used. SOD activities of all 158 isolates were measured and were divided into three groups: high-SOD activity (>0.22, n= 2), moderate-SOD activity (0.15≦≦0.22, n= 16) and low-SOD activity (<0.15, n= 140). Expressions of H. pylori Fe-SOD were examined by western blotting with anti-H. pylori Fe-SOD antibody prepared inhouse, and the profiles of Fe-SOD activity were investigated by zymogram with activity staining in native-PAGE. The characteristics of strains from high-SOD and low-SOD groups were examined under oxidative stress by paraquat. The average of H. pylori SOD activity was significantly higher in the cancer group than in the non-cancer group (P < 0.05). However, irrespective of SOD activity level, the amount of Fe-SOD expressed was variable among individual strains. Zymogram revealed a single band in moderate-SOD and low-SOD strains, but multiple bands in high-SOD strains were observed. These bands were confirmed as H. pylori Fe-SOD. Under oxidative stress with paraquat, low-SOD strains were drastically eliminated without inducible SOD activity; however, high-SOD strains were still viable with increased SOD activity. This study is the first to exhibit the characteristics of high-SOD activity strains representing multiple bands in zymograms and the correlation between H. pylori SOD activity and gastric cancer.     

Multiple Infection and Microdiversity among Helicobacter pylori Isolates in a Single Host in India

Helicobacter pylori is one of the most diverse bacterial species that chronically infects more than 70% of Indian population. Interestingly, data showing microdiversity of the H. pylori strains within a particular gastric niche remained scarce. To understand the extent of genetic diversity among H. pylori strains within a given host, 30 patients with gastro-duodenal problems were subjected to endoscopy and from each patient 10 single colonies were isolated. Characterization of each of these 10 single colonies by DNA fingerprinting as well as genotyping of several important genetic markers viz. cagA, vacA, iceA, vapD, cag PAI empty site, IS605, RFLP and two other genetic segments within cag PAI revealed that all of the 30 patients were infected with more than one strain and sometimes strains with 5 to 6 types of genetic variants. Analyses of certain genetic loci showed the microdiversity among the colonies from single patient, which may be due to the recombination events during long-term carriage of the pathogen. These results suggest that most of the patients have acquired H. pylori due to repeated exposure to this pathogen with different genetic make-up, which may increase the possibility of super infections. Genetic exchanges between these unrelated H. pylori strains may support certain H. pylori variant to grow better in a given host than the parental strain and thereby increasing the possibility for the severity of the infection.     

幽门螺杆菌随机扩增多态性DNA指纹图谱分析

应用随机扩增多态性DNA指纹法(RandomamplifiedpolymorphicDNA,RAPD)对来自29例消化性溃疡和19例单纯性胃炎病人的幽门螺杆菌(Helicobacterpylori,Hp)菌株基因组DNA进行PCR反应,建立Hp的DNA指纹图谱,运用统计分析软件(Statisticanalysissoftware,SAS)对HpDNA指纹图的相似性以及与疾病的相关性进行分析。结果显示,这些菌株按HpDNA指纹图可分为两大类,两种来源的菌株在两大类中的比例有明显差异(P<0.05)。提示可能存在与疾病相关的特异基因型。     

Diagnosis of Helicobacter pylori infection and determination of clarithromycin resistance by fluorescence in situ hybridization from formalin-fixed, paraffin-embedded gastric biopsy specimens

A reliable diagnostic test for Helicobacter pylori is important in clinical practice and research. The ideal diagnostic test for H. pylori should be sensitive, specific, and cost-effective. Helicobacter pylori resistance to clarithromycin is a common reason for failure of eradication therapy. The aim of this study was to evaluate the fluorescent in situ hybridization (FISH) method to detect H. pylori and determine clarithromycin resistance in formalin-fixed, paraffin-embedded gastric biopsy specimens. One hundred seventeen gastric biopsy specimens from patients with dyspepsia were examined for the presence of H. pylori by conventional culture, FISH, and histopathological methods. A set of fluorescent-labeled oligonucleotide probes binding to either H. pylori 16S rRNA or 23S rRNA sequences were used for FISH analysis. Phenotypic antibiotic susceptibilities of the isolates were tested using the Epsilometer test method (E test). Helicobacter pylori was detected in 70 of 117 biopsy specimens by histopathological examination and FISH, whereas it was detected in 47 specimens by culturing. Histopathology and FISH techniques failed to identify H. pylori in 1 biopsy sample isolated by culture. Clarithromycin resistance was found in 11 of 46 H. pylori isolates using the E test method. All of the phenotypic resistance measurements of isolates were correlated with genotypic clarithromycin resistance. Eleven clarithromycin-resistant strains were identified by FISH. The diagnosis of H. pylori infection and the determination of clarithromycin resistance in formalin-fixed, paraffin-embedded specimens using FISH is promising because it provides a rapid, reliable, and culture-independent diagnosis.     

Specific and sensitive detection of H. pylori in biological specimens by real-time RT-PCR and in situ hybridization

PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.     

IgG antibody titer against Helicobacter pylori correlates with presence of cytotoxin associated gene A-positive H. pylori strains

The level of the IgG antibody titer against Helicobacter pylori correlates with the severity of gastritis. H. pylori strains can harbor the so-called pathogenicity island, containing the cytotoxin associated gene (cagA). Since cagA-positive strains are more virulent it can be postulated that the gastritis will be more severe and hence the IgG antibody titer higher. In a cross-sectional study the correlation of IgG antibody titer and cagA status was studied from patients undergoing upper gastrointestinal endoscopy. Biopsy specimens were obtained to determine the H. pylori status. In addition a serum sample was taken for detection of IgG antibodies against H. pylori as well as CagA. A total of 290 patients positive for IgG antibodies against H. pylori were included. Of these 153 were cagA-positive and 137 were cagA-negative. The mean IgG antibody titer was significantly higher in cagA-positive patients compared to cagA-negatives, 0.75 (S.D. 0.22) versus 0.69 (S.D. 0.24) (P=0.033). It is concluded that the IgG antibody titer is significantly higher in patients harboring cagA-positive H. pylori strains. However, in daily practice the level in IgG antibody titer cannot predict whether or not an individual carries a cagA-positive H. pylori strain since major overlap in IgG antibody titer between cagA-positive and cagA-negative patients is present.     

Genetic variation among European strains of Saccharomyces paradoxus: results from DNA fingerprinting

We used microsatellite fingerprinting and RAPD analysis to characterize 28 wild European strains of Saccharomyces paradoxus. In contrast to our results from a previous allozyme survey [Naumov et al. Int. J. Syst. Bacteriol. 47: 341-344 (1997a)], these methods revealed extensive genetic variation. The RAPD primers 5'AATCGGGCTG and 5'GGGTAACGCC and the microsatellite primer (GTG)5 yielded reproducible amplification patterns of sufficient clarity and variability to distinguish individual strains from the wild. UPGMA analysis tended to group the strains according to climatic and geographic origin. A comparative study of Ty1 sequence having multiple chromosomal location was also done. Each wild S. paradoxus isolate shows a unique hybridization pattern allowing discrimination to the strain level.     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.     

Detection of Escherichia coli O157:H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses.

Twenty-five and three strains of Escherichia coli O157:H7 were identified from 25 tenderloin beef and three chicken meat burger samples, respectively. The bacteria were recovered using the immunomagnetic separation procedure followed by selective plating on sorbitol MacConkey agar and were identified as E. coli serotype O157:H7 with three primer pairs that amplified fragments of the SLT-I, SLT-II and H7 genes in PCR assays. Susceptibility testing to 14 antibiotics showed that all were resistant to two or more antibiotics tested. Although all 28 strains contained plasmid, there was very little variation in the plasmid sizes observed. The most common plasmid of 60 MDa was detected in all strains. We used DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) to compare the 28 E. coli O157:H7 strains. At a similarity level of 90%, the results of PFGE after restriction with XbaI separated the E. coli O157:H7 strains into 28 single isolates, whereas RAPD using a single 10-mer oligonucleotides separated the E. coli O157:H7 strains into two clusters and 22 single isolates. These typing methods should aid in the epidemiological clarification of the E. coli O157:H7 in the study area.     

Identification of mixed genotypes in Helicobacter pylori from gastric biopsy tissue by analysis or urease gene polymorphisms

Abstract PCR-RFLP analysis of the urease A and B genes was used to investigate the frequency that Helicobacter pylori from gastric biopsy specimens comprised genotypically distinct strains. Seventy-five strains of H. pylori from 44 subjects from four geographically diverse locations were examined and 31 distinct urease gene profiles were identified. For most cultures, the totals of the RFLP sizes were within 4% of the 2.41-kb PCR product. Five strains were genomically more complex suggesting they contained a mixture of related genotypes. The validity of the test was confirmed by analysis of a known strain mixture and of single colony picks from biopsy cultures. Urease gene PCR-RFLP profiling was shown to be more sensitive than ribotyping for determining the genotypic homogeneity of H. pylori .