Typing of Salmonella enterica Serotype Paratyphi C Isolates from Various Countries by Plasmid Profiles and Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) of 61 Salmonella enterica serotype Paratyphi C isolates from six countries gave five distinct clusters. Twenty-four isolates from five countries were susceptible to 10 antimicrobials tested and gave similar restriction endonuclease digest patterns of the 38-MDa plasmid. In contrast, plasmid and PFGE profiles of 37 multidrug-resistant isolates from Zaire were different from those from other countries.

Subgroup Determination of Bulgarian Isolates of Cucumber Mosaic Virus and the Presence of Satellite RNAs

Cucumber mosaic virus (CMV) is one of the most important viruses in Bulgaria, causing severe losses to agriculture, but little is known about the occurrence and distribution of subgroups within the country or the presence of satellite RNAs (satRNAs). Samples showing typical symptoms (mild to severe mosaic, vein clearing, vein necrosis, leaf deformation, stunting, and fruit necrosis) on several important crops (tomato, cucumber, pepper, bean, courgette, and tobacco) were collected from the main agricultural regions of the country. Isolates were maintained by sap inoculation in tobacco plants. Total RNAs were isolated from 38 samples (including two from bean) and used in reverse transcription-polymerase chain reaction (RT-PCR) assay with primers corresponding to the coat protein (CP) gene of RNA3 (3). A single strong band, 870 bp in length, was produced from all these samples. Amplified products were analyzed for subgroup differentiation by digestion with the restriction endonucleases MspI (3), PvuII, and EcoRI. The MspI subgroups 2 and 1 designated by Rizos et al. (3) according to their restriction endonuclease digest data correspond to the subgroups I and II widely used in the literature and based on serology, sequence data, and other properties. In this report, the subgroups are referred to as I and II for the sake of clarity. Isolates in both subgroups were found in all the main regions of Bulgaria. A few variations in MspI and EcoRI digestion patterns were seen, indicating some variability between isolates within subgroups. Only five samples, three from tomato and two from pepper, were found to be subgroup II. Subgroup I isolates were found in all the crops sampled. The PCR product from one representative isolate of each subgroup was cloned and sequenced by standard procedures. Alignment of the nucleotide and predicted amino acid sequences with published sequences of the CMV CP gene confirmed that the amplified products were derived from CMV. A further eight samples from bean gave only weak amplification and digestion of the products suggested they were likely to be subgroup II. However, these samples were unusual in not inducing symptoms in inoculated tobacco and in being difficult to propagate. The nature of these virus isolates is therefore unclear. Only a single occurrence in Bulgaria of satRNA of CMV has been reported (4) but in this study satRNAs were detected by RT-PCR (1) in total plant RNA extracts of 21 of the 38 samples tested. Amplified products of two of them, NB and 146D, were sequenced; comparison with published sequences confirmed that they were derived from CMV satellite. As expected from the symptoms induced by these isolates, a sequence homologous to the domain of satRNA Y responsible for bright yellow mosaic on tobacco (2) was identified in satRNA NB but not in satRNA 146D. satRNAs were not detected in the eight bean samples that had given only weak amplification with the CMV CP gene primers. The results presented here clearly demonstrate the presence of both subgroups of CMV in Bulgaria. Although CMV in Bulgaria has been studied previously by serological methods, no evidence had been found for the presence of subgroup II. References: (1) F. Grieco et al. Virology 229:166, 1997. (2) C. Masuta and Y. Takanami. Plant Cell 1:1165, 1989. (3) H. Rizos et al. J. Gen. Virol. 73:2099, 1992. (4) E. Stoimenova. J. Cult. Collect. 1:45, 1995.

Ribosomal RNA Gene Restriction Fragment Diversity amongst Penner Serotypes of Campylobacter jejuni and Campylobacter coli

Diversity based on ribosomal RNA gene-restriction endonuclease digest patterns was detected amongst forty-seven strains of Campylobacter made up of 38 strains of Campylobacter jejuni and 9 strains of Campylobacter coli. Restriction digests of chromosomal DNA prepared by treating with Hae III were probed with an oligonucleotide specific for Campylobacter 16S ribosomal RNA genes. Seventeen distinct hybridization patterns, each indicating the presence of 2 - 4 copies of the 16S rRNA gene are encoded in Campylobacter DNA. Differences in fragment patterns were observed not only between members of two species, but also between individual strains of the same species. Ribopattern fragments of 8.71, 7.56, 2.81 and 1.0 kb were characteristic of the majority of C. jejuni, whereas 7.59 and 4.68 kb fragments were commonly present in C. coli.In conclusion, Hae III ribotyping was even more discriminatory than the Penner serotyping of C. jejuni and C. coli, as strains of the same serotype were distinguished.

Restriction endonuclease characterization of resistant plasmids in Enterobacteriaceae isolated from children in the Sudan

SUMMARYThe investigation of plasmid similarity is an important component in the surveillance of antimicrobial resistance and in the detection of epidemic plasmids. The use of restriction endonucleases in the classification of transferable, multiply-resistant plasmids from faecal Enterobacteriaceae isolated at the Children's Emergency Hospital, Khartoum was investigated. Twenty-four transconjugant plasmids, coding for 11 different resistance patterns, each of molecular weight 62 MDa. were studied using four restriction enzymes;PstI,EcoR I,HindIII andAraII. Fifteen different digest profiles were obtained. Restriction profiles discriminated between plasmids with differing resistance patterns and demonstrated homology of plasmids with common resistance patterns. Restriction endonuclease digest patterns provide a potentially rapid and reproducible method of plasmid classification, that could contribute towards surveillance systems in tropical countries with a high prevalence of antimicrobial resistance.