Mapping the restriction fragments of the Brucella melitensis 16M genome with a new restriction endonuclease, Pacl, which cut the DNA into only eight fragments, indicated that this species contains two unique and independent replicons of about 2,100 and 1,150 kb. Pulsed-field gel electrophoresis of intact DNA revealed two bands migrating the expected distances. These replicons were identified as two unique and independent chromosomes by the presence of rRNA operons and genes for heat shock proteins mapping to separate replicons.Brucella melitensis is a species of the genus Brucella, members of which are small gram-negative bacteria that are pathogenic in many animals and occasionally in humans. The genetics of Brucella spp. are completely unknown. No nutritional mutants have been isolated and characterized because of the fastidious growth of these species and the difficulty in studying their strictly aerobic metabolism. In addition, chromosome exchange, by transformation, conjugation, or transduction, is completely unknown in these bacteria. Despite numerous attempts, plasmids or temperate bacteriophages have never been demonstrated in these species. Consequently, physical mapping of the Brucella genome was the first step to be undertaken in the study of Brucella genetics. In a previous paper (1), we concluded that this genome is a unique 3.1-megabase circular chromosome on which several genes are located. The map was obtained by the method described for mapping the Haemophilus influenzae genome (11). The Brucella genomic DNA was digested with three restriction endonucleases (SpeI, XhoI, and XbaI) and subjected to pulsed-field gel electrophoresis (PFGE), and the fragments obtained with one endonuclease were used as linking probes to join the fragments generated with the other two enzymes. However, we encountered several difficulties during the mapping, because no restriction endonuclease that cuts Brucella DNA (which has a G+C content of 59%) into less than 25 fragments was then known. It was therefore necessary to order a total of 113 Spel, XhoI, and XbaI fragments, a process resulting in some ambiguities in the map. Two new endonucleases are now available that recognize 8-bp sites on DNA containing only adenine and thymine and expected to be rare in high-G+C-content DNAs: Pacl (5'-TTAATTAA) and Swal (5'-AIT TAAAT). We used PacI in this study to

Twelve cases of infections caused by extended-spectrum beta-lactamase (ESBla)-producing Klebsiella pneumoniae were reported between August 1991 and March 1993 in the Geriatric Department of the Nimes University Hospital, where these bacteria had not been previously isolated. Restriction profiles of total genomic DNAs cleaved byXMal and Spel were compared by pulsed-field gel electrophoresis. The strains that were tested included the 12 isolates from K. pneumoniae-infected patients, strains recovered from rectal swabs of asymptomatic patients in the same ward, and strains isolated in other hospitals in Nimes at the same time. The restriction profiles of the 12 isolates and those recovered from asymptomatic patients in the same ward were very similar. Over a period of more than 1 year, extended-spectrum beta-lactamases were not detected in K. pneumoniae isolates with restriction patterns different from that of the epidemic strain. It seems, therefore, that there was no transfer of a plasmid or a gene coding for ESBla to strains of K. pneumoniae that were different from the epidemic strain. At the same time, ESBla-producing K. pneumoniae isolates exhibiting restriction endonuclease proffles very different from that of the epidemic strain were isolated from other hospitals in Nimes. None of these strains caused an outbreak. Pulsed-field gel electrophoresis, which allows precise characterization of strains beyond the species level, is a useful tool for studying the ESBla-producing K. pneumoniae strains involved in nosocomial outbreaks.

Epidemiological investigations of bacterial infections are generally based on multiple phenotypic markers that are often difficult to verify. A more general and reliable method is genomic DNA analysis by restriction endonucleases. However, the commonly used endonucleases produce too many fragments for correct separation by agarose electrophoresis. In contrast, simple electrophoretic patterns are obtained after genomic DNA digestion by low-frequency-cleavage restriction endonucleases and pulsed-field gel electrophoresis, making it easier to compare numerous strains from the same species. This technique was used to investigate an Acinetobacter calcoaceticus outbreak in a urologic department and bronchial colonization of artificially ventilated patients by Pseudomonas aeruginosa in an intensive care unit. The method allowed a clear distinction between epidemic and self-contaminating strains in these different epidemiological situations.

Genomic DNAs from taxonomically and epidemiologically well-defined strains of Acinetobacter baumannii were digested with restriction endonucleases that cleave with low frequency, and the fragments were separated by pulse-field gel electrophoresis. Restriction fragment length polymorphisms were observed. Restriction fragment length polymorphism analysis can be used as an epidemiological tool to delineate outbreaks of nosocomial infections caused by A. baumannii.