Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars.

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.

Characterization, distribution, and localization of ISRl2, and insertion sequence element isolated from Rhizobium leguminosarum bv. viciae.

An insertion sequence (IS) element, ISR12, from Rhizobium leguminosarum bv. viciae strain MSDJ4184 was isolated by insertional inactivation of the sacRB gene of pSUP104-sac, which allows positive selection. ISRl2 is 932 bp long, is flanked by 17-bp imperfect terminal inverted repeats, and generated a 3-bp target site duplication. ISRl2 was found to be 63 to 77% homologous to insertion elements of the IS5 group of the IS4 superfamily. A probe incorporating a full-length copy of ISRl2 was used to screen genomic DNAs from a collection of strains and from two field populations of R. leguminosarum to detect and estimate the copy numbers of homologous sequences. Among the collection of 63 strains representing the different species and genera of members of the family Rhizobiaceae, homology to ISRl2 was found within strains belonging to Sinorhizobium meliloti and S. fredii; within four of the six recognized Rhizobium species. R. leguminosarum, R. tropici, R. etli, and R. galegae; and within Rhizobium sp. (Phaseolus) genomic species 2. The apparent copy numbers of ISRl2 varied from one to eight. Among 139 isolates of R. leguminosarum from two field populations, homology to ISRl2 was detected in 91% of the isolates from one site and in 17% from the other. Analysis of the 95 isolates that hybridize to ISRl2 revealed a total of 20 distinct hybridization patterns composed of one to three bands. Probing blots of Eckhardt gels showed that sequences with homology to ISRl2 may be found on plasmids or the chromosome. Analysis of their genomic distribution demonstrated relationships and diversity among the R. leguminosarum isolates tested.

Typing of Listeria strains by random amplification of polymorphic DNA.

The polymerase chain reaction (PCR) was used to obtain randomly amplified polymorphic DNA (RAPD) profiles for typing of Listeria strains. In this procedure, whole cells were incubated in the reaction mixture. The discriminating ability of a randomly designed 10-mer primer, HLWL74, was assessed. A total of 60 collection strains of Listeria, encompassing all 7 Listeria species and all known serovars was submitted to PCR with the primer HLWL74. Upon agarose gel electrophoresis, 29 different banding profiles were reproducibly obtained. No common profiles were recorded for strains from different Listeria species. For various groups of strains sharing the same serotype (e.g. 4b, 1/2a, 1/2b), RAPD analysis could generate further subdivision. On the other hand, some strains from different serotypes produced identical RAPD profiles with the primer HLWL74. The RAPD typing method from whole cells is proposed as an attractive alternative for other Listeria typing systems, and the 10-mer HLWL74 as a primer to include in a forthcoming set of standard primers for RAPD typing of Listeria isolates.

A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemiological studies of Listeria monocytogenes isolates.

The analysis of RAPD profiles generated by PCR with a single 10-mer, HLWL74, was compared to bacteriophage susceptibility data for epidemiological typing of Listeria monocytogenes strains. A total of 104 L. monocytogenes strains was screened, all from serogroup 1 or serotype 4b. Of these, 53 had been isolated during 6 different listeriosis outbreaks. The remaining 51 strains were chosen randomly from our collection. A total of 38 RAPD types were observed, although each epidemic group of strains isolated during one of these outbreaks displayed a specific RAPD profile. For 98% of the strains isolated during outbreaks, the correlation between RAPD typing and phage typing was complete. Only one strain, typed as epidemic by phage typing, was clearly distinguishable from the others by RAPD analysis. Among the 51 strains not related to an outbreak, 12 were linked to epidemic groups by RAPD analysis. Two of these rearrangements were supported by phage typing. The remaining 10 strains could be excluded by phage typing from any of the epidemic groups studies. Considering all 104 isolates, the decision to relate a strain to a particular epidemic group or to exclude a strain from any epidemic group was the same for 92 isolates, using either phage typing or RAPD analysis. The RAPD analysis, which is quick, simple and suited for automation, is proposed as an attractive alternative for phage typing in epidemiological studies of listeriosis.

Study on the epidemiology and control of Campylobacter jejuni in poultry broiler flocks.

Broiler flocks are frequently infected with Campylobacter jejuni. The origin of the infection is still unclear. The question of whether colonization of flocks results from transmission of C. jejuni from breeder flocks to progeny (vertical transmission) or from environmental sources (horizontal transmission) remains to be answered. Therefore, in this study samples were taken from successive broiler flocks in two broiler houses (house A on farm A and house B1 on farm B) as well as from the environment of the houses. All C. jejuni isolates were typed by using the Penner serotyping system, and part of the isolates from farm B were typed by using a randomly amplified polymorphic DNA-typing system. In poultry house A, C. jejuni was isolated from the first flock but not from subsequent flocks. In poultry house B1, C. jejuni strains of the same Penner serotypes and exhibiting identical DNA profiles were isolated from successive flocks. Infection of the flocks from a common source via horizontal pathways is suspected, while a vertical route of infection is not likely to exist. Application of measures to control horizontal transmission of C. jejuni on farm B was successful.