Analysis of genetic variability,antimicrobial susceptibility and virulence markers in Helicobacter pylori identified in Central Italy

Objective. To assess the relationship between the presence of mixed infection of Helicobacter pylori and both antimicrobial susceptibility and virulence markers. Material and methods. Thirty-six patients with H. pylori infection were included in the study. Three colonies were selected from each positive biopsy sample collected from each host for a total of 108 H. pylori strains. The genetic variability was evaluated through the amplified fragment length polymorphism (AFLP) analysis; the antibiotic susceptibility to amoxicillin, clarithromycin, moxifloxacin, rifabutin and tinidazole was determined using the minimum inhibitory concentrations (MICs) with the agar dilution method. Moreover, the vacA, cagA, iceA and babA2 statuse were detected by polymerase chain reaction (PCR). Results. There was a strong connection between mixed H. pylori infection and antimicrobial resistance. In particular, H. pylori strains with genetic variability, in the same host, expressed more resistance to clarithromycin, moxifloxacin and tinidazole than that expressed in strains with a unique genetic host pattern. VacA s1m1/s1m2 genotypes were found in 70% of strains isolated in mixed infection, whereas the same allelic combinations were found in 42% of strains, isolated in single infection. The cagA⁺ status prevailed both in patients with mixed (97%) and in those with single infection (85%) without significant differences. The iceA1 status was more commonly found in patients with mixed infection, whereas the babA2 status was significantly prevalent in single H. pylori infection. Conclusions. Mixed H. pylori infection harbouring in one patient is significantly related to strains that are more resistant to antibiotics and with a more virulent genotype (vacA s1m1/s1m2, cagA, iceA1) than strains responsible for single infection.     

A hospital-associated outbreak of Legionnaires' disease caused by Legionella pneumophila serogroups 4 and 10 with a common genetic fingerprinting pattern

An outbreak of eight cases of pneumonia caused by Legionella pneumophila non-serogroup 1 (non-sg 1) occurred at a Swedish university hospital in 1993. Including previous and subsequent sporadic cases, the total number of culture-positive patients was 13. Twelve available non-sg1 isolates from patients were compared to 50 environmental water isolates using a monoclonal antibody test for serogrouping and amplified fragment length polymorphism analysis (AFLP). Of the 12 hospital-associated Legionella non-sg 1 patient isolates, 4 were serogrouped as sg 4, 7 as sg 10, and one as sg 6. Using AFLP fingerprinting all serogroup (sg) 4 and 10 isolates were genetically related except for minor variations. Furthermore, sg 4 isolates were identical in AFLP to sg 10 isolates. Patient isolates were also identical to isolates found in the water system of several hospital buildings, but quite unrelated to isolates obtained in a subsequent outbreak at the same hospital caused by L. pneumophila sg 1. Serogroup variations in outbreaks may occur despite a common molecular fingerprinting pattern. Evidently, the L. pneumophila sg 4 and 10 strains were closely related genetically, which raises the question whether this variation in phenotype is due to a genetic event or to a variable phenotypic expression. Genetic fingerprinting should be used in conjunction with serogrouping in epidemiological investigations.     

An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110 restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting

A rapid, simple and highly discriminatory DNA fingerprinting methodology which produces data that can be easily interpreted, compared and transported is the ultimate goal for studying the epidemiology of Mycobacterium tuberculosis. A novel TaqI fluorescent amplified fragment length polymorphism (fAFLP) approach to M. tuberculosis DNA fingerprinting that targeted the variable IS6110 marker was developed in this study. The new method was tested for specificity and reproducibility, and compared with the standard reference IS6110 restriction fragment length polymorphism (RFLP) method for a panel of 78 isolates. Clustering conflicts between the two methods were resolved using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) data. Comparison with an in-silico digestion of strain H37Rv showed that fAFLP-detected fragments were highly specific in vitro. The reproducibility of repeated digestions of strain H37Rv was 100%. Clustering results obtained by fAFLP and RFLP were highly congruent, with fAFLP allocating 97% of RFLP-clustered isolates to the same eight clusters as RFLP. Two single-copy isolates that had been clustered by RFLP were not clustered by fAFLP, but the MIRU-VNTR patterns of these isolates were different, indicating that the RFLP data had falsely clustered these isolates. Analysis by fAFLP will allow rapid screening of isolates to confirm or refute epidemiological links, and thereby provide insights into the frequency, conservation and consequences of specific transposition events.     

AFLP allows the identification of genomic markers of ruminant Chlamydia psittaci strains useful for typing and epidemiological studies

Amplified fragment length polymorphism (AFLP), a novel method for molecular typing, was evaluated for its ability to differentiate among a group of highly related Chiamydia psittaci strains isolated from ruminants and belonging to serotype 1. A total set of 12 strains were included in this study, 10 strains inducing abortion in ruminants and 2 strains from faecal samples. For the AFLP analysis, the total purified genomic DNA of each strain was submitted to a one-step digestion-ligation reaction for 3 h at 37°C. DNA was digested with a single restriction endonuclease Mspl and ligated to specially constructed adapters. Subsequently, restricted fragments were selectively amplified under high stringency PCR conditions using primers complementary to the adapters. Amplified products were then resolved on agarose gel electrophoresis. The method is easy to perform, fast and reproducible. AFLP enabled characterization of C. psittaci strains at the infra-subspecific level. Thus, AFLP led to the identification of a cluster of strains on the basis of their AFLP patterns, constituted by French chlamydial isolates. It also permitted differentiation among strains in relation to host origin and to clinical syndromes. These data confirmed the highly discriminative power of AFLP towards the differentiation of closely related ruminant C. psittaci strains. The analysis will need to be applied to more samples to check the usefulness of AFLP markers in epidemiological and evolutionary studies.     

Amplified fragment length polymorphism (AFLP) analysis is useful for distinguishing Leishmania species of visceral and cutaneous forms

The Leishmania strains belonging to cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) have been reported to possess close homology in genome profiles. To confirm this on genetic basis an attempt was made to differentiate Leishmania major; Leishmania tropica and Leishmania donovani genetically for the first time using amplified fragment length polymorphism (AFLP)—a high throughput DNA fingerprinting technique. The objective of this research work was to identify DNA markers of CL and VL. Ten combinations of selective primers detect a total of 1487 informative AFLP marker. Percentage of polymorphism was 45.12%. Three hundred and thirty-seven unique AFLP markers were also identified in three species of Leishmania. A clear distinction was revealed between L. major and L. donovani. It was inferred by AFLP analysis that a higher rate of polymorphisms occurred among Leishmania species which indicate the distinguished pattern of the disease cause by Leishmania, i.e. VL and CL. Analysis based on polymorphic AFLP markers revealed considerably high genetic variation among the genome of these species which was sufficient to distinguish between CL and VL.     

半夏栽培品遗传差异的AFLP分析

目的构建不同地区来源半夏栽培品的DNA指纹图谱,探讨利用AFLP分子标记在半夏遗传多样性、亲缘关系及种质鉴别上的可行性。方法运用扩增片段长度多态性技术(AFLP),对我国17个地区的51份半夏栽培品和4份掌叶半夏样品(外群)进行基因组DNA多态性分析。结果从64对引物组合中筛选出8对多态性丰富、鉴别效率高的AFLP引物组合,构建了51份半夏栽培品的AFLP指纹图谱。聚类分析结果表明:所有半夏栽培品完全被区分开,来源地相同的种质表现出相对密切的亲缘关系,浙江等华东地区的栽培半夏同其他地区半夏有着明显遗传差异,聚类分析结果获得了Bootstrap校验的支持。结论AFLP分子标记可用于半夏遗传多样性、亲缘关系及种质鉴别分析,浙江、江苏等华东地区栽培半夏的遗传特性相对独立。     

金铁锁种质资源的遗传多样性分析

目的研究中国西南特有濒危药用植物金铁锁的遗传多样性。方法应用AFLP分子标记技术对具有代表性的7个金铁锁居群,共137个个体进行分析。结果在金铁锁的物种水平上,Nei’s基因多样性指数(He)、Shan-non’s信息指数(I)、多态位点百分率(PPB)分别为0.2434±0.1791、0.3735±0.2485、82.30%;在其居群水平上分别为0.0918±0.1610、0.1402±0.2362、30.48%。居群间Nei遗传分化指数(Gst):0.6244;Shannon’s多样性基因遗传分化系数(Ist):0.6246。聚类表明:地理距离相对远的丽江居群与个旧居群遗传距离最近;昆明居群与其他居群遗传距离较远。统计获得9条可区分各地方居群的特征指纹带。结论金铁锁种内的遗传多样性水平丰富,而居群内遗传多样性水平较低,居群间遗传分化显著;各居群间亲缘关系与地理距离无明显相关性;其种内特征带与各居群特征带结合,可以为AFLP分子标记技术用于其种质资源的鉴定、遗传育种提供重要的依据。     

扩增片段长度多态性技术对淋球菌的基因分型

目的：评价扩增片段长度多态性技术(AFLP)分析对淋球菌分离株进行基因分型的能力。方法：26株淋球菌分离株基因以EcoRI和MesⅠ酶切及AFLP分析。结果：同一地区的淋球菌分离株之间存在相当大的DNA多态性。结论：AFLP是鉴别淋球菌临床分离株有用而敏感的分子技术，有助于了解菌株来源、菌株间的克隆相关性及抗生素耐药性的传播。