F. Muñoa

Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: Relationship with expression of adhesins and production of toxins**

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesin-encoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap+ and/or sfa+ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing alpha-haemolysin (Hly+) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesin-encoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.

Escherichia coli hha mutants, DNA supercoiling and expression of the haemolysin genes from the recombinant plasmid pANN202-312

The hha gene of Escherichia coli was identified as modulating the expression of the haemolysin (hly) genes encoded by the recombinant plasmid pANN202‐312. hha mutants harbouring plasmid pANN202‐312 showed increased haemolysin production. The product of the hha gene, the Hha protein, shows strong homology to the YmoA protein of Yersinia enterocolitica, which plays a role in the thermoregulation of various V. enterocolitica virulence genes. We show in this study that the Hha protein modulates the expression of haemolysin at the transcriptional level in ceils harbouring plasmid pANN202‐312. In addition, hha mutants show alterations in the level of plasmid DNA supercolting. This suggests that the hha mutation increases haemolysin expression through changes in the DNA topology. This hypothesis is supported by our finding that gyr mutations, inhibitors of DNA gyrase such as novo‐biocin, or growth in conditions reported to reduce levels of negative supercoiling, such as low osmolarity medium, increase haemolysin production.

Complementation of the hha mutation in Escherichia coli by the ymoA gene from Yersinia enterocolitica : dependence on the gene dosage

The Hha protein from Escherichia coli is highly similar (82%) to the YmoA protein from Yersinia enterocolitica. Both are members of a new class of proteins that modulates gene expression, probably by influencing DNA topology. In this paper, complementation of the hha mutation in E. coli by the ymoA gene from Y. enterocolitica has been studied. We show that the ymoA gene complements one of the phenotypic properties of hha mutants (high level of haemolysin production when they carry the recombinant plasmid pANN202-312) when cloned in a medium-copy-number plasmid but not when carried in a low-copy-number plasmid. Western blot analysis of the expression of YmoA in E. coli rules out inefficient expression of the protein. Surprisingly, the hha gene itself fails to complement the hha mutation when cloned in a medium-copy-number vector and causes genetic rearrangements of the E. coli chromosome as a consequence of insertion sequences mobilization.

Influence of lipopolysaccharide on external hemolytic activity of Salmonella typhimurium and Klebsiella pneumoniae

Excretion ofEscherichia coli α-hemolysin was tested in rough and smooth strains ofKlebsiella pneumoniae andSalmonella typhimurium. Smooth strains harboring the hemolytic recombinant plasmid pANN202-312 showed a five- to tenfold increase in the hemolytic activity evaluated in the external medium compared with isogenic rough strains harboring the same plasmid.

Prevalence and Characteristics of Enteropathogenic Escherichia coli with the eae Gene in Diarrhoeic Rabbits

A field study was carried out with the objective of investigating the prevalence of enteropathogenic Escherichia coli (EPEC) with the eae gene in diarrhoeic rabbits. EPEC eae+ were isolated from 60 (74%) of 81 diarrhoeic rabbits sampled in 30 industrial fattening farms localized in the four provinces of Galicia (northwestern Spain). Attaching and effacing lesions were found in 44 of 50 animals processed for histology. The 111 E. coli strains identified belonged to 19 different O serogroups and 13 biotypes. However, 53 (48%) of the strains belonged to serogroup O103 and 36 (32%) showed the serobiotype O103:B14. The eae gene was significantly more frequent (100%; 47 of 47) among the highly pathogenic rhamnose‐negative strains of serobiotypes O103:B6 and O103:B14 than among the E. coli strains belonging to other serobiotypes (36%; 23 of 64) (P < 0.001). In this first report about the prevalence of EPEC with the eae gene in rabbits, we conclude that the class of E. coli strains observed is a common cause of diarrhoea in Galician rabbit farms, and that highly pathogenic rhamnose‐negative strains of serotype O103:K‐:H2 and biotype B14 are specially predominant.

Occurrence of secondary transpositions of Tn5 upon Tn5 mutagenesis inEscherichia coli

When Tn5 insertions were obtained in thehha gene ofEscherichia coli HB101 harboring the hemolytic multicopy plasmid pANN202-312, most of thehha mutants obtained that produced larger amounts of hemolysin than the wild-type cells segregated into 10 percent of clones, which did not further produce hemolysin. We demonstrate here that a secondary transposition of Tn5 intohlyA, the structural gene for hemolysin, was responsible for such phenotype.