Background: The EFLM Task and Finish Group Urinalysis has updated the ECLM European Urinalysis Guidelines (2000) on urinalysis and urine bacterial culture, to improve accuracy of these examinations in European clinical laboratories, and to support diagnostic industry to develop new technologies. Recommendations: Graded recommendations were built in the following areas: Medical needs and test requisition: Strategies of urine testing are described to patients with complicated or uncomplicated urinary tract infection (UTI), and high or low-risk to kidney disease. Specimen collection: Patient preparation, and urine collection are supported with two quality indicators: contamination rate (cultures), and density of urine (chemistry, particles). Chemistry: Measurements of both urine albumin and α1-microglobulin are recommended for sensitive detection of kidney disease in high-risk patients. Performance specifications are given for urine protein measurements and quality control of multiproperty strip tests. Particles: Procedures for microscopy are reviewed for diagnostic urine particles, including urine bacteria. Technologies in automated particle counting and visual microscopy are updated with advice how to verify new instruments with the reference microscopy. Bacteriology: Chromogenic agar is recommended as primary medium in urine cultures. Limits of significant growth are reviewed, with an optimised workflow for routine specimens, using leukocyturia to reduce less important antimicrobial susceptibility testing. Automation in bacteriology is encouraged to shorten turn-around times. Matrix assisted laser desorption ionization time-of-flight mass spectrometry is applicable for rapid identification of uropathogens. Aerococcus urinae, A. sanguinicola and Actinotignum schaalii are taken into the list of uropathogens. A reference examination procedure was developed for urine bacterial cultures.

Horizontal gene transfer is a key step in the evolution of bacterial pathogens. Besides phages and plasmids, pathogenicity islands (PAIs) are subjected to horizontal transfer. The transfer mechanisms of PAIs within a certain bacterial species or between different species are still not well understood. This study is focused on the High-Pathogenicity Island (HPI), which is a PAI widely spread among extraintestinal pathogenic Escherichia coli and serves as a model for horizontal transfer of PAIs in general. We applied a phylogenetic approach using multilocus sequence typing on HPI-positive and -negative natural E. coli isolates representative of the species diversity to infer the mechanism of horizontal HPI transfer within the E. coli species. In each strain, the partial nucleotide sequences of 6 HPI–encoded genes and 6 housekeeping genes of the genomic backbone, as well as DNA fragments immediately upstream and downstream of the HPI were compared. This revealed that the HPI is not solely vertically transmitted, but that recombination of large DNA fragments beyond the HPI plays a major role in the spread of the HPI within E. coli species. In support of the results of the phylogenetic analyses, we experimentally demonstrated that HPI can be transferred between different E. coli strains by F-plasmid mediated mobilization. Sequencing of the chromosomal DNA regions immediately upstream and downstream of the HPI in the recipient strain indicated that the HPI was transferred and integrated together with HPI–flanking DNA regions of the donor strain. The results of this study demonstrate for the first time that conjugative transfer and homologous DNA recombination play a major role in horizontal transfer of a pathogenicity island within the species E. coli. The species Escherichia coli comprises non-pathogenic, commensal bacterial strains belonging to the normal gut microbiota of humans and many animals, but also pathogenic strains, which cause different types of intestinal or extraintestinal infections in man and animals. Single factors and mechanisms involved in pathogenesis of extraintestinal pathogenic E. coli (ExPEC) have been analyzed in detail for many years. The genetic information of these virulence factors has largely been acquired by horizontal DNA transfer. Key elements of horizontal transfer are large DNA fragments, called genomic islands, integrated into the conserved E. coli chromosomal backbone. The transfer of genomic islands within the E. coli species, however, has yet been elusive. In this study, we focused on the High-Pathogenicity Island (HPI), which is a genomic island widely spread among E. coli. It serves as a model for horizontal transfer within the E. coli species. We used a combination of sequenced based methods (Multi Locus Sequence Typing) and DNA–transfer experiments to decipher the transfer mechanisms of DNA–islands within the E. coli species. The results of this study demonstrate for the first time that conjugative transfer and homologous DNA recombination play a major role in horizontal transfer and spread of a pathogenicity island within E. coli.

Infections due to extraintestinal pathogenicEscherichia coli(ExPEC) are common in humans and animals and include urinary tract infections (from uropathogenicE. coli[UPEC]), septicemia, and wound infections. These infections result in significant morbidity and mortality and in high health care costs. In view of the increasing number of ExPEC infections and the ever-growing antibiotic resistance capability of ExPEC isolates, preventive measures such as an effective vaccine against ExPEC are desirable. An ExPEC vaccine may be cost-effective for select patient groups. Previous vaccine candidates consisted of single target proteins or whole ExPEC cells. Here we describe a subunit vaccine against ExPEC which is based on immunodominant epitopes of the virulence-associated ExPEC proteins FyuA, IroN, ChuA, IreA, Iha, and Usp. Using a novel approach of computer-aided design, two completely artificial genes were created, both encoding eight peptide domains derived from these ExPEC proteins. The recombinant expression of these two genes resulted in a protein vaccine directed against ExPEC but not against commensalE. coliof the gut flora. In mice, the vaccine was highly immunogenic, eliciting both strong humoral and cellular immune responses. Nasal application resulted in high secretory immunoglobulin A (sIgA) production, which was detectable on the mucosal surface of the urogenital tract. Finally, it conveyed protection, as shown by a significant reduction of bacterial load in a mouse model of ExPEC peritonitis. This study provides evidence that a novel vaccine design encompassing distinct epitopes of virulence-associated ExPEC proteins may represent a means for providing a protective and pathogen-specific vaccine.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains possess several siderophore-dependent iron uptake systems. In this study we demonstrated that the salmochelin siderophore receptor IroN is involved in the invasion of urothelial cells by ExPEC in vitro. Thus, IroN may play a dual role in the establishment of urinary tract infections, displaying an iron uptake receptor as well as an internalization factor.