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The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC) of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC). Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC). After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H-, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF). The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H-, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized.

An analysis for stx(2) variants among the 2010 human stx(2)-positive Shiga toxin-producing Escherichia coli (STEC) strains from Germany collected at the National Reference Centre 1999-2008 revealed 0.6% to possess the recently described stx(2g) gene. Sequencing of the whole stx(2g) operons showed new alleles and pseudogenes. The further molecular, phenotypic, and phylogenetic comparison of 12 human stx(2g)-harbouring isolates with 12 stx(2g)-harbouring isolates from animals or environmental sources demonstrated that both groups are closely related, indicating the human infections as a potential zoonotic disease. Although originating from various different sources, the stx(2g)-containing strains belong to only 3 phylogenetic lineages, represented by 4 serovars belonging to 4 sequence types. In view of the huge diversity among other STEC, this suggests the emergence of the stx(2g) variant as a rather recent microevolutionary event. Interestingly, in the strains under investigation, Stx2g was not expressed. However, all of them contained the estIa gene which typically is associated with enterotoxin-producing E. coli and did express STIa. By this combination of virulence genes of different pathotypes of intestinal pathogenic E. coli, these strains represent a new, intermediate pathotype and emerging pathogens. Given a rising number of intermediate pathotypes becoming described among E. coli, a wider range of virulence markers should be included in the regular pathotype diagnostics., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Piglet pathogenic Escherichia coli encoding Shigatoxin 2e and F18 adhesins are the etiological agents of oedema disease as well as of non-oedema disease colibacillosis. In order to reveal virulence differences among this pathogen, the presence of the pathogenicity island (PAI) E. coli type three secretion system 2 (ETT2) was examined. Using PCR and Southern blot techniques for the identification of the right, the middle, and the left region of this 29.9kb large genetic element, the entire ETT2 was found among E. coli O138:H(-), O139:H1, and O147:H6 strains originated from cases of oedema disease in Germany between 1995 and 2001 and belonging to various clonal types. In contrast, non-oedema disease E. coli isolates (e.g. O8:H19, 101:H(-), O141:H4) contain deleted subtypes of ETT2. These deletions cover the translocon part of the putative ETT2-encoded type III secretion apparatus. It is suggested that the entire ETT2 is associated with a particular virulence trait of piglet oedema disease E. coli (EDEC).

Plasmids belonging to Escherichia coli incompatibility group Q are relatively small (approximately 5 to 15 kb) and able to replicate in a remarkably broad range of bacterial hosts. These include gram-positive bacteria such as Brevibacterium and Mycobacterium and gram-negative bacteria such as Agrobacterium, Desulfovibrio, and cyanobacteria. These plasmids are mobilized by several self-transmissible plasmids into an even more diverse range of organisms including yeasts, plants, and animal cells. IncQ plasmids are thus highly promiscuous. Recently, several IncQ-like plasmids have been isolated from bacteria found in environments as diverse as piggery manure and highly acidic commercial mineral biooxidation plants. These IncQ-like plasmids belong to different incompatibility groups but have similar broad-host-range replicons and mobilization properties to the IncQ plasmids. This review covers the ecology, classification, and evolution of IncQ and IncQ-like plasmids.

The analysis of the complete nucleotide sequence of the small resistance plasmid pIE1107 revealed a close similarity to the well-known IncQ plasmids. Highly conserved replication proteins and nearly identical origins of replication (oriV) suggest equivalent functions in the related replication systems. However, pIE1107 contains two copies of IncQ-oriV-like DNA which are slightly different regarding the iterons. Upon deletion of a silent copy of IncQ-oriV-like DNA the resulting plasmid is fully compatible with IncQ plasmids, indicating that there is no mutual communication between the replication control of the respective replicons. Experiments with cloned oriV DNA strongly suggest that the replication initiation protein of pIE1107 has specialized into the distinct target-iterons of its own oriV which differs only by a few nucleotides from the oriV of IncQ plasmids. Implications from the apparent highly specific protein-DNA recognition and from the incompatibility properties of pIE1107 for the evolution of a family of compatible, IncQ-like plasmids are discussed., (Copyright 1998 Academic Press.)

A 1,3 kb DNA fragment of the IncM plasmid R446, if cloned in a multicopy plasmid, inhibits in trans the expression of conjugation pili by IncM plasmid-harbouring host bacteria as indicated by their insensitivity to the IncM pilus-dependent bacteriophage phi M (Iml, insensitivity to phi M-mediated lysis). Determination of the nucleotide sequence of this DNA fragment, the introduction of deletions an the analysis of transposon insertions reveal two determinants, imlA and imlB, responsible for the Iml phenotype. A stretch of 80 bp of DNA containing imlA and about 450 bp of adjacent DNA comprising imlB, together, bring about inhibition of the typical expression of conjugation pili at 30 degrees C. The introduction of a transposable promoter probe and the construction of respective lacZ fusions indicate transcription of complementary strands in vivo overlapping in the region comprising imlA and imlB. Moreover, the expression of reporter genes discloses temperature-dependent transcription of the imlA-imlB-region in one direction. A particular subfragment of the 1.3 kb IncM plasmid-derived DNA does not inhibit conjugation pilus expression at 30 degrees C but stimulates in trans the formation of pili at 42 degrees C to give rise to untypical sensitivity to phi M at 42 degrees C in addition to 30 degrees C. Other subfragments reveal vital interferences with IncM plasmid-harbouring host cells. The putative nature of the cloned determinants interfering with the normal expression of IncM plasmid DNA is discussed.

The transposon Tn7 codes for a trimethoprim resistance and for a streptomycin/spectinomycin resistance function of the bacterial host cells. Cloning of a restriction fragment of Tn7 into the vector plasmid pUC19 reveals the presence in Tn7 of an additional potential resistance determinant. A streptothricin resistance gene, which appears cryptic in the original Tn7 context becomes activated in the recombinant plasmid upon supplying the promoter function of the lacZ system of pUC19. These results together with previously published sequence data further disclose the modular character in the resistance gene regions of Tn7-like transposons.

The biotin-labelled DNA probe for identification of functioning and silent genes for streptotricin acetylation has been constructed. The probe is homologous to sat1 gene of the movable genetic element Tn1825. The simplified modification of the hybridization technique using the biotin-labelled DNA probe is described.

To localize the insertion sites for Tn7-like transposons Tn1824, Tn1825 and Tn1826 the EcoRI-cleaved fragments of E. coli recA+ and recA- strains chromosome carrying the transposons were hybridized. It was shown that transposition of Tn7-like elements takes place in the strictly defined region of E. coli chromosome, like it had been previously described for transposon Tn7.

Fifty-nine Escherichia coli strains belonging to two clonal groupings were investigated for major outer membrane proteins, colicin production, and partly for plasmid DNA content. The membrane protein patterns of the 01:K1:H7(H-):F11 and O1:K1:H-:F9 strains obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were distinctly different from each other and, therefore, are useful for clonal assignment. All of the F11 isolates had one plasmid of about 85 Md in common which is suggested to be characteristic for the clone. Their content of smaller plasmid DNA was heterogeneous and showed geographical differences. The F9 strains showed a plasmid pattern different from the F11 strains. A plasmid of 2.6 Md was found in all of these isolates. Colicin production was found to be useful for clonal assignment only in two thirds of the F9 and not in the F11 strains. Some strains with identical properties seem to be of epidemic importance.

14 conjugative plasmids originated from different bacterial hosts and geographical sources were identified as members of the incompatibility group IncX with R6K as the reference plasmid. In spite of their close genetic relatedness (transfer and incompatibility properties), their molecular sizes ranged between 39 and 77 kb and DNA fingerprinting with endonucleases such as EcoRI, EcoRV, BamHI, BglI, BglII, PstI and SalI revealed a great variety of fragment patterns.

Plasmids, which code for a new type of drug resistance due to the acetylation of streptothricin and belonging to various incompatibility groups (I1, I2, W3, FII, S, X, and N) were identified in gram-negative bacteria. The gene for the acetyltransferase is closely linked to a gene for the streptomycin/spectinomycin adenylyltransferase AAD-3" on a novel type of a transposon, designated Tn1825. This is related to Tn7.

New IncQ R plasmids, pIE639 and pIE723, are characterized and compared to the prototype IncQ plasmid RSF1010. Additional resistance determinants not common on other R plasmids are located on small stretches of DNA interspacing essential regions at different positions in an otherwise unchanged core of IncQ plasmid DNA. The contribution of IncQ plasmids to resistance evolution in bacteria is discussed.

Natural isolates of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Providencia stuartii were analysed to determine their plasmid content. This data allowed the identification of nosocomial strains of K. pneumoniae and P. stuartii and helped in the differentiation of epidemic strains of E. coli 0111 and S. typhimurium. Phenotypically similar isolates of S. typhimurium could be shown to be of independent origin using plasmid pattern analysis. The dissemination of a particular plasmid through different strains of S. typhimurium resulted in a simulation of a very widely distributed epidemic strain, because the plasmid interfered with the phage type of its host strain in addition to determining resistance properties. Plasmid pattern analysis disclosed two independently existing but interacting epidemic processes: a bacterial 'epidemic' strain may become disseminated over a large territory and may predominate there for a long time; a single plasmid, however, may also become distributed through many different bacterial strains and may spread over a large territory. Plasmid pattern analysis provides a valuable and universal epidemiological laboratory method.