Húgyúti kórokozó Escherichia coli virulenciájára irányuló vizsgálatok = Investigations on the virulence of uropathogenic Escherichia coli

Az Escherichia coli fő húgyúti virulencia faktorainak vizsgálatával bizonyítottuk, hogy az alfa hemolizin a toxicitásért felelős legjelentősebb pathogenetikai tényező, emellet a tok és fimbriák a kolonizációban jászanak fontos szerepet. A haemolysin operont tartalmazó pathogenitási szigetet in vitro és egerek bélscatornájában in vivo is át tudtuk vinni a genus más képviselőjébe. Emellett a hemolizin operon kicserélhetőségét bizonyítottuk Escherichia és Proteus törzsek között szintén in vivo környezetben is. A hemolizáló rekombinánsok virulenciája a recipienshez képest szignfikánsan magasabb. Hibridizációs kísérletekkel az E. coli és Proteus penneri haemolizin operonjainak homológiáját bizonyítottuk. Gyakorlati jelentőségű megfigyelés, hogy a rezisztens hemolizáló törzs antibiotikum kezelés esetén a bélcsatornában elszaporodva életet veszélyeztető fertőzés forrása lehet. A hemolzin és fimbria operonok szabályozásában jelenős mechanizmusokat tártunk fel. | In mouse experiments we have proved that alpha-haemolysin with its toxic effect is the main urinary virulence factor of E. coli. Besides fimbrial adhesins and the capsule are important in the first colonisation step of the infection. The pathogenicity island containing the haemolysin operon could be transferred from the wild type strain to non-hemolytic E. coli strains in the mouse intestine. Furthermore, the haemolysin operon is exchangable in vivo between E. coli and Proteus strains. The haemolytic recombinants present with significantly increased virulence in various mouse models. With hybridisation experiments we could prove homology between the haemolysin operons of E. coli and Proteus penneri. The observation that antibiotic resistant haemlysin producing E. coli in the microbiota may elicit severe infection in the antibiotic treated patients may be of paractical significance. Regulatory machineries governing the expression of haemolysin and fimbria operons were also elucidated

Effect of primycin on growth-arrested cultures and cell integrity of Staphylococcus aureus

Bactericidal effect against non-dividing bacteria is a very advantageous, but rare characteristic among antimicrobial agents, mostly possessed by those affecting the cell membrane. These kinds of agents can kill bacterial cells without lysis. We assessed these characteristics on primycin, a topical anti-staphylococcal agent highly effective against prevalent multiresistant strains, as it also acts on the cell membrane. In time-kill studies, primycin preserved its bactericidal activity against growth-arrested Staphylococcus aureus cultures. The bactericidal action was slower against growth-arrested cultures compared to the exponentially growing ones to different extents depending on the manner of arrest. The bactericidal effect was less influenced by stringent response and by protein synthesis inhibition, proving that it does not depend on metabolic activity. In contrast, uncoupling of the membrane potential predominantly slowed, and low temperature almost stopped killing of bacteria. In consideration of published data, these facts suggest that the antibacterial action of primycin involves disrupting of the membrane potential, and is predominantly influenced by the membrane fluidity. Optical density measurements and transmission electron microscopy verified that primycin kills bacterial cells without lysis. These results reveal favorable characteristics of primycin and point to, and broaden the knowledge on its membrane-targeted effect

Globális regulátor mutációknak mint az attenuálás lehetőségének vizsgálata Escherichia coli-ban = Investigation on global regulator mutations as possible tools of attenuation in Escherichia coli

Az Escherichia coli globális regulátor génjeinek a virulencia funkciókra irányuló hatását vizsgálva megállapítottuk, hogy a húgyúti fertőzést és újszülöttkori meningitist okozó törzsek esetében a leuX és az rfaH gének kiesése (utóbbi Salmonella enterica esetében is) attenuált származékokat eredményez, a recA ás lrhA gének a virulenciát mérhetően nem befolyásolják. Tárgyaljuk a regulátoroknak az egyes virulencia faktorok expressziójára gyakorolt hatását. Az attenuálás olyan fontos virulencia funkciók kiesésére vezethető vissza, mint a haemolysin termelés, a tokantigén képzése, vagy a komplett "O" antigén szintézise. A származékok enterális kolonizáló képessége is csökken, ami a fenti gének szerepére utal abban, hogy az E. coli a kommenzális bélflóra részeként az extraintestinalis fertőzések forrásaként rezervoár szerepet töltsön be. A kutatások perspektivikusan vakcina jelölt származékok kifejlesztését eredményezhetik. | Investigations on the influence of global regulatory factors on Escherichia coli virulence have lead to the conclusion that leuX and rfaH (the latter also in the case of Salmonella enterica) play a pivotal role in pathogenic capacity. On the other hand knock out recA and lrhA mutants presented with equal virulence to the wild type. Effect of the regulators on the function of individual virulence factors is discussed: attenuation of virulence is due to downregulation of such important attributes like haemolysin production, capsule synthesis and expression of a complete 'O' antigen. Handicapped intestinal colonization capacity of the derivatives points to an important role for these regulators in sustaining the reservoir function of intestinal commensals as potential pathogens in subsequent extraintestinal manifestations. Our data imply a perspective for development of potential vaccine candidate derivatives

Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic Escherichia coli in vitro support the role of conjugation for horizontal transfer of genomic islands

<p>Abstract</p> <p>Background</p> <p>A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs) - including pathogenicity islands (PAIs) - in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far.</p> <p>Results</p> <p>To study mobilisation of such large genomic regions in prototypic uropathogenic <it>E. coli </it>(UPEC) strain 536, PAI II₅₃₆was supplemented with the <it>mob</it>_RP4region, an origin of replication (<it>oriV</it>_<it>R6K</it>), an origin of transfer (<it>oriT</it>_<it>RP4</it>) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II₅₃₆construct occured from strain 536 into an <it>E. coli </it>K-12 recipient. In transconjugants, PAI II₅₃₆existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient's chromosome at the <it>leuX </it>tRNA gene. This locus is the chromosomal integration site of PAI II₅₃₆in UPEC strain 536. From the <it>E. coli </it>K-12 recipient, the chromosomal PAI II₅₃₆construct as well as the CIs could be successfully remobilised and inserted into <it>leuX </it>in a PAI II₅₃₆deletion mutant of <it>E. coli </it>536.</p> <p>Conclusions</p> <p>Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands.</p

Identification and characterization of CTX-M-15 producing Klebsiella pneumoniae clone ST101 in a Hungarian university teaching hospital

We investigated the molecular epidemiology of extended spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae isolates derived from the teaching hospitals of University of Pécs, Pécs, Hungary in the time period 2004–2008. Molecular typing, antimicrobial susceptibility testing, detection of common β-lactamase genes (blaCTX-M, blaTEM and blaSHV) and virulence associated traits (hypermucoviscosity, magA, k2a, rmpA, siderophores, type 1 and 3 fimbria, biofilm formation, serum resistance) were performed for 102 isolates. The results showed the presence of three major ciprofloxacin resistant CTX-M-15 producing clones (ST15 n = 69, ST101 n = 10, and ST147 n = 9), of which ST15 was predominant and universally widespread. Considering distribution in time and place, ST101 and ST147 were detected at fewer inpatient units and within a narrower time frame, as compared to ST15. Beside major clones, eleven minor clones were identified, and were shown to harbour the following β-lactamase genes: six clones carried blaCTX-M, four clones harboured blaSHV-5 and one clone possessed both blaCTX-M and ESBL type blaSHV. Among the SHV-5 producing K. pneumoniae clones a novel sequence type was found, namely ST1193, which harboured a unique infB allele. Different virulence factor content and peculiar antimicrobial susceptibility profile were characteristic for each clone. In contrast to major clone isolates, which showed high level resistance to ciprofloxacin, minor clone isolates displayed significantly lower MIC values for ciprofloxacin suggesting a role for fluoroquinolones in the dissemination of the major K. pneumoniae clones. This is the first description of the CTX-M-15 producing K. pneumoniae clone ST101 in Hungary

Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic Escherichia coli in vitro support the role of conjugation for horizontal transfer of genomic islands

Background: A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs) - including pathogenicity islands (PAIs) - in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far. Results: To study mobilisation of such large genomic regions in prototypic uropathogenic E. coli (UPEC) strain 536, PAI II536 was supplemented with the mobRP4 region, an origin of replication (oriVR6K), an origin of transfer (oriTRP4) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II536 construct occured from strain 536 into an E. coli K-12 recipient. In transconjugants, PAI II536 existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient’s chromosome at the leuX tRNA gene. This locus is the chromosomal integration site of PAI II536 in UPEC strain 536. From the E. coli K-12 recipient, the chromosomal PAI II536 construct as well as the CIs could be successfully remobilised and inserted into leuX in a PAI II536 deletion mutant of E. coli 536. Conclusions: Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands

Virulence Traits of Inpatient Campylobacter jejuni Isolates, and a Transcriptomic Approach to Identify Potential Genes Maintaining Intracellular Survival

There are still major gaps in our understanding of the bacterial factors that influence the outcomes of human Campylobacter jejuni infection. The aim of this study was to compare the virulence-associated features of 192 human C. jejuni strains isolated from hospitalized patients with diarrhoea (150/192, 78.1%), bloody diarrhoea (23/192, 11.9%), gastroenteritis (3/192, 1.6%), ulcerative colitis (3/192, 1.5%), and stomach ache (2/192, 1.0%). Traits were analysed with genotypic and phenotypic methods, including PCR and extracellular matrix protein (ECMP) binding, adhesion, and invasion capacities. Results were studied alongside patient symptoms, but no distinct links with them could be determined. Since the capacity of C. jejuni to invade host epithelial cells is one of its most enigmatic attributes, a high throughput transcriptomic analysis was performed in the third hour of internalization with a C. jejuni strain originally isolated from bloody diarrhoea. Characteristic groups of genes were significantly upregulated, outlining a survival strategy of internalized C. jejuni comprising genes related (1) to oxidative stress; (2) to a protective sheath formed by the capsule, LOS, N-, and O- glycosylation systems; (3) to dynamic metabolic activity supported by different translocases and the membrane-integrated component of the flagellar apparatus; and (4) to hitherto unknown genes

Type 1 Fimbriae, a Colonization Factor of Uropathogenic Escherichia coli, Are Controlled by the Metabolic Sensor CRP-cAMP

Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues