Variable properties among Escherichia coli isolates include serotype, electrophoretic migration of major outer membrane proteins, metabolic properties, production of hemolysin or colicin or both, and plasmid content. These characteristics were compared in E. coli strains of capsular types Kl, K5, K92, and K100 and in non-encapsulated isolates. The 234 bacterial strains from the United States and Europe which we studied had been isolated from healthy or diseased individuals recently or as long ago as 1941. Regardless of source, most 07:K1, 016:K1, and 075:K100 isolates could be assigned to three unique, serotypespecific groups, which were interpreted as representing three bacterial clones. Two bacterial (sub)clones each were discerned among the 018:K1 and 018:K5 isolates, and two further, distinct clones were discerned among the 01:K1 isolates. The implications of these results for epidemiological analyses and for MATERIALS AND METHODS Bacterial strains. Bacterial strains isolated recently in several countries were obtained from the following individuals:

The identification and speciation of strains ofLegionella is often difficult, and even the more successful chromatographic classification techniques have struggled to discriminate newly described species. A sequence-based genotypic classification scheme is reported, targeting approximately 700 nucleotide bases of the mip gene and utilizing gene amplification and direct amplicon sequencing. With the exception ofLegionella geestiana, for which an amplicon was not produced, the scheme clearly and unambiguously discriminated among the remaining 39 Legionella species and correctly grouped 26 additional serogroup and reference strains within those species. Additionally, the genotypic classification of approximately 150 wild strains from several continents was consistent with their phenotypic classification, with the exception of a few strains where serological cross-reactivity was complex, potentially confusing the latter classification. Strains thought to represent currently uncharacterized species were also found to be genotypically unique. The scheme is technically simple for a laboratory with even basic molecular capabilities and equipment, if access to a sequencing laboratory is available.

The contribution of two unrelated Aeromonas hydrophila fihaemolytic toxins to virulence was assessed in a suckling mouse model. The first haemolysin gene, isolated from an A. hydrophila A6 cosmid bank, encoded a potential gene product of 621 amino acids and a predicted molecular size of 690 kDa. The inferred amino acid sequence showed 89% identity to the AHHI haemolysin of A. hydrophila ATCC 7966, and 51 O/ O identity to the HlyA haemolysin of Vibrio cholerae E l Tor strain 017. The second haemolysin gene (designated aerA), which encodes aerolysin, a pore-forming toxin, was partially cloned by PCR for the purpose of mutant construction. This PCR product was a 1040 bp fragment from the C-terminal region of aerA. It is proposed that the 69.0 kDa V. cholerae-HlyA-like haemolysin gene be termed hlyA to contrast with the aerA terminology for the aerolysin. A suicide vector was used to inactivate both the hlyA and aerA genes in A. hydrophila A6. When assessed in the suckling mouse model, only the hlyA aerA double mutant showed a statistically significant reduction in virulence -a 20-fold change in LD,, (Scheffe test, P < 0 0 5 ) . Cytotoxicity t o buffalo green monkey kidney cell monolayers and haemolysis on horse blood agar were eliminated only in the hlyA aerA double mutants. This is the f i r s t report of cloning and mutagenesis of two unrelated haemolytic toxin genes in the same strain of a mesophilic aeromonad. For A. hydrophila, a two-toxin model provides a more complete explanation of virulence.

Multilocus sequence typing (MLST) is a relatively new high-resolution typing system employed for epidemiological studies of bacteria, including Salmonella. Discrimination based on MLST of housekeeping genes may be problematical, due to the high identity of gene sequences of closely related Salmonella species. The presence of genomic sequences derived from stable temperate phages in Salmonella offers an alternative for MLST of Salmonella. We have used MLST of prophage loci in Salmonella enterica serovar Typhimurium to discriminate closely related isolates of serovar Typhimurium. We have compared these results to MLST of five housekeeping genes, as well as pulsed-field gel electrophoresis (PFGE). The presence or absence of prophage loci in the 73 serovar Typhimurium isolates tested, as well as allelic variation as detected by sequencing, provided greater discrimination between isolates than either MLST of housekeeping genes or PFGE. Amplification of prophage loci alone separated serovar Typhimurium isolates into 27 groups comprising multiple isolates or individual strains. Sequencing of isolates found within the clusters separated isolates even further. By contrast, PFGE could only divide the 73 isolates into five distinct groups. MLST using housekeeping genes did not provide any significant separation of isolates in comparison to amplification or MLST of prophage loci. The results demonstrate that the amplification and sequencing of prophage loci provides a high-resolution, objective method for the discrimination of closely related isolates of serovar Typhimurium. It is proposed that multiple amplification of phage locus typing may provide sufficient discrimination for epidemiological purposes without recourse to MLST.

Previous studies have shown that two hemolytic toxins, HlyA and AerA, contribute to the virulence of Aeromonas hydrophila. A survey was performed to gauge the distribution of hlyA and aerA genes in clinical and environmental Aeromonas isolates. For A. hydrophila, A. veronii biotype sobria and A caviae, 96%, 12% and 35% of strains, respectively, were hlyA positive, whereas, 78%, 97%, 41%, respectively, were aerA positive. All virulent A. hydrophila isolates were hlyA aerA . This genotype was most common in A. hydrophila (75.4%) followed by A. caviae (29.4%) and A. veronii biotype sobria (9.6%). For A. hydrophila, a two-hemolytic toxin model of virulence provides the best prediction of virulence in an animal model. z