Antiadhesive hydroalcoholic extract from Apium graveolens fruits prevents bladder and kidney infection against uropathogenic E. coli.

Fruits from Apium graveolens (Celery) are used traditionally in Persian and European medicine for the treatment of uncomplicated urinary tract infections. No data are available on A. graveolens extract on the interplay between uropathogenic E. coli and the eukaryotic host cells and on quorum sensing of the bacteria. The present study aimed to characterize an antiadhesive and anti quorum sensing effect of a characterized A. graveolens extract by specific in vitro assays and to correlate these effects with in vivo data obtained by an animal infection model. Hydroalcoholic extract CSE (EtOH-water, 1:1) from A. graveolens fruits was characterized by UHPLC/+ESI-QTOF-MS and investigated on antiproliferative activity against UPEC (strain NU14) and human T24 bladder cells. Antiadhesive properties of CSE were investigated within two different in vitro adhesion assays. For in vivo studies BALB/c mice were used in an UPEC infection model. The effect of CSE on bacterial load in bladder tissue was monitored within a 4- and 7 days pretreatment (200, 500 mg/kg) of the animals. CSE was dominated by the presence of luteolin-glycosides and related flavons besides furocoumarins. CSE had no cytotoxic effects against UPEC and bladder cells. CSE exerts a dose dependent antiadhesive activity against UPEC strains NU14 and UTI89. CSE inhibited in a concentration-dependent manner bacterial quorum sensing. 4- and 7-day pretreatment of animals with CSE transurethrally infected with UPEC NU14, significantly reduced the bacterial load in bladder tissue. CSE is assessed as an antiadhesive extract for which the traditional use in phytotherapy for UTI is justified.

Aqueous extract from Orthosiphon stamineus leaves prevents bladder and kidney infection in mice.

BACKGROUND: Extracts from the leaves of Orthosiphon stamineus are used in phytotherapy for treatment of uncomplicated urinary tract infections. PURPOSES: Evaluation of an aqueous extract against infection with uropathogenic Escherichia coli in vivo; investigation of underlying microbiological mechanisms. STUDY DESIGN: In vivo studies in mice and in vitro investigations on cytotoxicity, antiadhesive potential, influence on bacterial gene expression and quorum sensing. METHODS: Extract OWE was prepared by hot water extraction. For in vivo studies BALB/c mice were used in an UPEC infection model. The effect of OWE on bacterial load in bladder/kidney tissue was monitored in pre- and posttreatment. Cytotoxicity of OWE against different UPEC strains, T24 bladder/A498 kidney cells, gene expression analysis, monitoring of phenotypic motility and quorum sensing was investigated by standard methods of microbiology. RESULTS: OWE was quantified (UHPLC) according to the content of rosmarinic acid, cichoric acid, caffeic acid. Three- and 5-day treatment of animals with OWE (750mg/kg) after transurethral infection with UPEC CFT073 reduced the bacterial load in bladder and kidney, similar to norfloxacin. Four- and 7-day pretreatment of mice prior to the infection with UPEC NU14 reduced bacterial bladder colonization. In vitro investigations indicated that OWE (≤2mg/ml) has no cytotoxic or proliferation-inhibiting activity against different UPEC strains as well as against T24 bladder and A498 kidney cells. OWE exerts a dose dependent antiadhesive activity against UPEC strains NU14 and UTI89. OWE reduced gene expression of fimH, but evoked increase of the expression of motility/fitness gene fliC. Increase of bacterial motility on gene level was confirmed by a changed bacterial phenotype by an increased bacterial motility in soft agar assay. OWE inhibited in a concentration-dependent manner bacterial quorum sensing. CONCLUSION: OWE is assessed as a strong antiadhesive plant extract for which the traditional use in phytotherapy for UTI might be justified.

Escherichia coli: an old friend with new tidings.

Escherichia coli is one of the most-studied microorganisms worldwide but its characteristics are continually changing. Extraintestinal E. coli infections, such as urinary tract infections and neonatal sepsis, represent a huge public health problem. They are caused mainly by specialized extraintestinal pathogenic E. coli (ExPEC) strains that can innocuously colonize human hosts but can also cause disease upon entering a normally sterile body site. The virulence capability of such strains is determined by a combination of distinctive accessory traits, called virulence factors, in conjunction with their distinctive phylogenetic background. It is conceivable that by developing interventions against the most successful ExPEC lineages or their key virulence/colonization factors the associated burden of disease and health care costs could foreseeably be reduced in the future. On the other hand, one important problem worldwide is the increase of antimicrobial resistance shown by bacteria. As underscored in the last WHO global report, within a wide range of infectious agents including E. coli, antimicrobial resistance has reached an extremely worrisome situation that 'threatens the achievements of modern medicine'. In the present review, an update of the knowledge about the pathogenicity, antimicrobial resistance and clinical aspects of this 'old friend' was presented.

[Evolution and infection biology of hemolytic-uremic syndrome (HUS) associated E. coli (HUSEC)]. / Evolution und Infektionsbiologie der mit dem hämolytisch-urämischen Syndrom (HUS) assoziierten E. coli (HUSEC).

Shiga toxin (Stx)-producing Escherichia coli (STEC), which cause hemolytic-uremic syndrome (HUS), are designated as HUSEC. Their exceptional genome variability driven by evolutionary diversification permits fast adaptation to changed environmental conditions. The HUSEC collection (http://www.ehec.org), which has been established at the Institute for Hygiene in Münster, contains 42 EHEC reference strains (HUSEC001-HUSEC042). It represents a unique repository collection of pathogens and is extremely helpful for the analysis of evolutionary changes and fixed properties in the STEC that cause the most severe host injury. Such genomic attributes include slowly evolving loci, mobile genetic elements that often encode virulence factors and are assimilated via horizontal gene transfer. Current evolutionary models indicate that numerous outbreak strains evolved recently and that highly pathogenic HUSEC descend from less pathogenic progenitors. However, additional data suggest that HUSEC have small effective population sizes. The HUSEC collection is also a valuable resource with which to study important non-Shiga toxin virulence factors.

[Virulence factors of uropathogens]. / Virulenzfaktoren uropathogener Erreger.

In contrast to many commensal bacteria, uropathogens are equipped with many virulence-associated factors that facilitate infection of different areas of the urinary tract. Different extra- and intracellular bacterial lifestyles result in acute or recurrent urinary tract infection. Adhesins promote adhesion or invasion as well as biofilm formation. Flagella do not only allow ascension from the urethra to the bladder and even further to the kidneys, but can also function as adhesins and invasins. In later stages of an infection, toxins may destroy host cells, thus protecting against the host response or releasing nutrients. Extracellular polysaccharides, capsules, and the O-antigen protect bacteria against the host immune system. Furthermore, uropathogens may also directly inhibit the mucosal inflammatory response. Specific metabolic traits promote fitness, growth and survival in the urinary tract.

Adaptation of Pathogenic E. coli to Various Niches: Genome Flexibility is the Key.

It is a well-known observation and a long-standing hypothesis that pathogen genome dynamics are important in infectious disease processes. Recent achievements in large-scale genome sequencing, comparative genomics and molecular epidemiology help to unravel current challenges of E. coli pathogenomics, i.e. to gain insights into the in vivo relevance of genome dynamics. Data from comparative genomics support the hypothesis of widespread involvement of horizontal gene transfer in the evolution of E. coli, leading to the presence of distinct and variable 'genomic islands' within the conserved 'chromosomal backbone' in several bacterial lineages. Extensive gene acquisition and loss provide different lineages with distinct metabolic, pathogenic and other capabilities. Not only mobile genetic modules but also point mutations facilitate rapid adaptation of E. coli to changing environmental conditions and hence extend the spectrum of sites that can be infected. We report on recent research efforts to analyze pathoadaptive and other genomic alterations of the E. coli genome that affect disease severity and may have consequences for diagnostics and treatment of E. coli infections.

The molecular basis of infectious diseases: pathogenicity islands and other mobile genetic elements. A review.

Bacterial genomes generally consist of stable regions termed core genome, and variable regions that form the so-called flexible gene pool. The flexible part is composed of bacteriophages, plasmids, transposons as well as unstable large regions that have been termed genomic islands. Genomic islands encoding virulence factors of pathogenic bacteria have been designated "pathogenicity islands". Pathogenicity islands were first discovered in uropathogenic Escherichia coli and presently more than 30 bacterial species carrying pathogenicity islands have been described. This review summarises the current knowledge on bacterial genomic islands and their general features, and discusses their putative role in the evolution of microbes in the light of genomics of pathogenic bacteria.

Pathogenicity islands of uropathogenic E. coli and the evolution of virulence.

Uropathogenic Escherichia coli (UPEC) are the most important group of microorganisms responsible for urinary tract infection. UPEC differ from non-pathogenic E. coli and from other E. coli pathotypes by the production of specific virulence factors, which enable the bacteria to adhere to uroepithelial cells and to establish urinary tract infections. Besides adherence factors, toxins, 'modulins', capsules, iron uptake systems and other bacterial products contribute to the virulence of the strains. The respective genes are frequently located on large pieces of DNA called 'pathogenicity islands' (PAIs). PAIs form (unstable) regions of the genome of UPECs, which are often associated with tRNA genes. Using various molecular techniques, the functions of PAI encoded gene products have been studied. The usage of DNA arrays give answers to questions on the distribution of PAIs among various enterobacteria and on the expression of the different genes under in vitro and in vivo conditions. In addition, assumptions can be made on the evolution of these important pathogens.

Efficient expression of the alpha-haemolysin determinant in the uropathogenic Escherichia coli strain 536 requires the leuX-encoded tRNA(5)(Leu).

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries two alpha-haemolysin determinants which are located on different pathogenicity islands (PAI I(536) and PAI II(536)). PAI II(536) is associated with the tRNA gene leuX. The leuX-encoded tRNA(5)(Leu) is required for the efficient expression of the hly determinants in strain 536. HlyA levels were reduced and secretion of the protein was delayed in the leuX-negative mutant strain 536Delta102. The lack of a functional tRNA(5)(Leu) resulted in a decrease in hly transcript levels in comparison to the wild-type strain. Analysis of several genes whose products are involved in the regulation of hly expression revealed that levels of RfaH and Hha, as well as the corresponding rfaH and hha transcripts, were higher in the leuX-negative background, whereas the expression of tolC and hns was not influenced by the leuX genotype. The analysis of hly transcript levels in hha deletion mutants of the E. coli strains 536 and 536Delta102 demonstrated that the increase in hha expression is partially responsible for the reduction in hly transcript levels in the leuX-negative background. These results demonstrate that the tRNA(5)(Leu) affects the expression of the alpha-haemolysin determinant at different levels in a regulatory cascade, and imply that, in addition to Hha, at least one further, as yet unidentified, regulatory factor must be involved in the regulation of hly transcription in the uropathogenic E. coli strain 536.

Whole genome plasticity in pathogenic bacteria.

The exploitation of bacterial genome sequences has so far provided a wealth of new general information about the genetic diversity of bacteria, such as that of many pathogens. Comparative genomics uncovered many genome variations in closely related bacteria and revealed basic principles involved in bacterial diversification, improving our knowledge of the evolution of bacterial pathogens. A correlation between metabolic versatility and genome size has become evident. The degenerated life styles of obligate intracellular pathogens correlate with significantly reduced genome sizes, a phenomenon that has been termed "evolution by reduction". These mechanisms can permanently alter bacterial genotypes and result in adaptation to their environment by genome optimization. In this review, we summarize the recent results of genome-wide approaches to studying the genetic diversity of pathogenic bacteria that indicate that the acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content.

Identification and characterization of a novel genomic island integrated at selC in locus of enterocyte effacement-negative, Shiga toxin-producing Escherichia coli.

The selC tRNA gene is a common site for the insertion of pathogenicity islands in a variety of bacterial enteric pathogens. We demonstrate here that Escherichia coli that produces Shiga toxin 2d and does not harbor the locus of enterocyte effacement (LEE) contains, instead, a novel genomic island. In one representative strain (E. coli O91:H(-) strain 4797/97), this island is 33,014 bp long and, like LEE in E. coli O157:H7, is integrated 15 bp downstream of selC. This E. coli O91:H(-) island contains genes encoding a novel serine protease, termed EspI; an adherence-associated locus, similar to iha of E. coli O157:H7; an E. coli vitamin B12 receptor (BtuB); an AraC-type regulatory module; and four homologues of E. coli phosphotransferase proteins. The remaining sequence consists largely of complete and incomplete insertion sequences, prophage sequences, and an intact phage integrase gene that is located directly downstream of the chromosomal selC. Recombinant EspI demonstrates serine protease activity using pepsin A and human apolipoprotein A-I as substrates. We also detected Iha-reactive protein in outer membranes of a recombinant clone and 10 LEE-negative, Shiga toxin-producing E. coli (STEC) strains by immunoblot analysis. Using PCR analysis of various STEC, enteropathogenic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, uropathogenic E. coli, and enteroinvasive E. coli strains, we detected the iha homologue in 59 (62%) of 95 strains tested. In contrast, espI and btuB were present in only two (2%) and none of these strains, respectively. We conclude that the newly described island occurs exclusively in a subgroup of STEC strains that are eae negative and contain the variant stx(2d )gene.

Regulation of tRNA5Leu-encoding gene leuX that is associated with a pathogenicity island in the uropathogenic Escherichia coli strain 536.

Pathogenicity islands (PAls) contain virulence determinants and are often linked to tRNA loci. The leuX gene encoding tRNA5Leu is associated with PAI II536 of the uropathogenic E. coli strain 536 (O6:K15:H31) and is important for the expression of virulence factors in this strain. Transcription of leuX differs from that of the majority of tRNA genes including the major leucyl-tRNA gene leuV. Availability of the tRNAsLeu can be linked with the efficient expression of virulence determinants. Transcription of leuX and letV was studied to gain further insights into the regulation of this tRNA gene and its role during the expression of virulence determinants. leuX but not letV transcripts accumulated in stationary phase. While leuX transcription was unchanged or slightly up-regulated by an increase in growth temperature, osmolarity or ethanol concentration, that of leuV was reduced. The heat shock-specific sigma factor RpoH was shown to affect transcription of leuX but not of leuV. These results demonstrate that transcription of a tRNA gene can be specifically influenced by an alternative sigma factor. Our finding expands our knowledge of the regulation of tRNA gene transcription and represents an additional mode of regulation of gene expression that can also affect bacterial virulence.

S-Fimbria-encoding determinant sfa(I) is located on pathogenicity island III(536) of uropathogenic Escherichia coli strain 536.

The sfa(I) determinant encoding the S-fimbrial adhesin of uropathogenic Escherichia coli strains was found to be located on a pathogenicity island of uropathogenic E. coli strain 536. This pathogenicity island, designated PAI III(536), is located at 5.6 min of the E. coli chromosome and covers a region of at least 37 kb between the tRNA locus thrW and yagU. As far as it has been determined, PAI III(536) also contains genes which code for components of a putative enterochelin siderophore system of E. coli and Salmonella spp. as well as for colicin V immunity. Several intact or nonfunctional mobility genes of bacteriophages and insertion sequence elements such as transposases and integrases are present on PAI III(536). The presence of known PAI III(536) sequences has been investigated in several wild-type E. coli isolates. The results demonstrate that the determinants of the members of the S-family of fimbrial adhesins may be located on a common pathogenicity island which, in E. coli strain 536, replaces a 40-kb DNA region which represents an E. coli K-12-specific genomic island.

A subtractive hybridisation analysis of genomic differences between the uropathogenic E. coli strain 536 and the E. coli K-12 strain MG1655.

Suppression subtractive hybridisation (SSH) was performed to identify genomic differences between the uropathogenic Escherichia coli strain 536 and the non-pathogenic E. coli K-12 strain MG1655. In total, 22 DNA fragments were isolated which were specific for strain 536. Five of these fragments showed homology to known virulence determinants and four fragments matched genes for lipopolysaccharide (LPS) or capsule biosynthesis and a siderophore receptor. Seven fragments did not show any homology to known genes. These fragments may represent parts of putative pathogenicity islands (PAIs). Whereas two fragments were highly specific for uropathogenic E. coli (UPEC), the other fragments could also be detected among the other tested wild-type strains.

Expression of hemin receptor molecule ChuA is influenced by RfaH in uropathogenic Escherichia coli strain 536.

The outer membrane protein ChuA responsible for hemin utilization has been recently identified in several pathogenic Escherichia coli strains. We report that the regulatory protein RfaH influences ChuA expression in the uropathogenic E. coli strain 536. In an rfaH mutant, the chuA transcript as well as the ChuA protein levels were significantly decreased in comparison with those in the wild-type strain. Within the chuA gene, a consensus motif known as the JUMPStart (just upstream of many polysaccharide associated gene starts) sequence was found, which is shared by RfaH-affected operons. Furthermore, the presence of two different subclasses of the chuA determinant and their distribution in E. coli pathogroups are described.

Toxin genes on pathogenicity islands: impact for microbial evolution.

Toxin-specific genes are often located on mobile genetic elements such as phages, plasmids and pathogenicity islands (PAIs). The uropathogenic E. coli strain 536 carries two alpha-hemolysin gene clusters, which are part of the pathogenicity islands I536 and II536, respectively. Using different genetic techniques, two additional PAIs were identified in the genome of the E. coli strain 536, and it is likely that further PAIs are located on the genome of this strain. Pathogenicity islands are often associated with tRNA genes. In the case of the E. coli strain 536, the PAI-associated tRNA gene leuX, which encodes a minor leucyl-tRNA, affects the expression of various virulence traits including alpha-hemolysin production. The exact mode of action of the tRNA5Leu-dependent gene expression has to be identified in the future.