Monika Kerényi

Virulence factors of uropathogenic Escherichia coli

Virulence factors of Escherichia coli are of two main types; those produced on the surface of the cell and those produced within the cell and then exported to the site of action. Those on the surface include different sorts of fimbriae that have a role in adhesion to the surface of host cells but may also have additional roles such as tissue invasion, biofilm formation or cytokine induction. The activities of cell wall components are discussed and several exported virulence factors are described that have anti host cell activities. Others virulence factors enable the bacteria to grow in an environment of iron restriction.

TRNA GENES AND PATHOGENICITY ISLANDS : INFLUENCE ON VIRULENCE AND METABOLIC PROPERTIES OF UROPATHOGENIC ESCHERICHIA COLI

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries two unstable DNA regions on its chromosome which were termed pathogenicity islands (Pais). Both pathogenicity islands, Pai I and Pai II, are incorporated into tRNA specific loci: Pai I is located in the tRNA gene for selenocysteine (selC), and Pai II is integrated in the leucine‐specific tRNA locus leuX. Mutant strain 536−21 has lost the two pathogenicity islands together with the intact tRNA genes. While 536 is a virulent strain, 536−21 has lost a number of properties, including in vivo virulence. In previous publications we reported that the genes coding for two haemolysins (hly I, hly II) and P‐related fimbria (prf) are located on the Pais. In this paper, we demonstrate that the expression of other gene products influencing metabolic properties in addition to in vivo virulence are strongly dependent on the intact tRNA loci selC and leuX. In order to determine the influence of the two tRNAs on the expression of these properties, the genes seIC and leuX were cloned from the genome of strain 536 and then introduced into the mutant 536−21. Our results clearly show that the selenocysteine‐specific tRNA (tRNASec) directly influences the ability of the bacteria to grow under anaerobic conditions, because selenocysteine is part of the enzyme formate dehydrogenase (FDH) which is involved in mixed acid fermentation. The rare leucine‐specific tRNA5Leu, encoded by leuX, influences a number of properties including type 1 fimbria production, flagellation and motility, production of enterobactin and serum resistance, and is also necessary for full in vivo virulence. While the tRNASec is directly involved in the production of FDHs, the leuX specific tRNA5Leu appears to influence the expression of various factors through specific transcriptional or translational control mechanisms.

Occurrence of hlyA and sheA Genes in Extraintestinal Escherichia coli Strains

ABSTRACT The association of a hemolytic phenotype with the carriage of the α-hemolysin gene (hlyA) and/or the silent hemolysin gene (sheA or clyA) among 540 extraintestinal clinical isolates of Escherichia coli and 110 fecal isolates from healthy individuals was investigated. Though HlyA is an important virulence factor in extraintestinal E. coli infection, the role of SheA is not completely clarified. Two hemolytic sheA+E. coli strains that lacked hlyA and possessed no other hemolysin genes were identified. No hlyA+sheA+ strains were identified, suggesting that there is possible incompatibility between hlyA and sheA in the chromosome of E. coli.

Bacterial growth in ropivacaine hydrochloride.

IMPLICATIONS Drugs affecting bacterial growth may influence the occurrence of postoperative infections. Ropivacaine 10 mg/mL killed Staphylococcus aureus and Escherichia coli; ropivacaine 2 mg/mL supported the growth of E. coli.

A comparison of the antimicrobial property of lidocaine/prilocaine cream (EMLA®) and an alcohol-based disinfectant on intact human skin flora.

BACKGROUND: The application of EMLA® cream is indicated for topical anesthesia of the skin in connection with IV cannulation. Recently, we described that EMLA cream has an antibacterial effect in vitro. METHODS: The impact of the local anesthetic lidocaine/prilocaine cream (EMLA) on intact human skin flora was compared to that of an alcohol-based skin disinfectant (Skinsept Pur®). Samples were taken from 0 to 12 h after treatment. RESULTS: The number of colony forming units (cfu) on the skin decreased significantly after both EMLA and Skinsept Pur treatment from 44.9 ± 1.3 (42.4 ± 7.0) to 0.9 ± 0.17 (1.61 ± 0.47) cfu/cm2, respectively (mean ± sem), at the first sampling time (1 h) and remained significantly below 0 h values for the study period. The cfu count was significantly lower with EMLA cream at 4, 6, and 12 h compared to Skinsept Pur. CONCLUSION: EMLA cream has a longer bacteriostatic effect after early bactericidal impact compared to skin disinfection with Skinsept Pur.

Diversity among clinical isolates of Proteus penneri detected by random amplified polymorphic DNA analysis

DNA of thirteen haemolytic Proteus penneri strains of clinical origin, all producing calcium dependent haemolysin and having been derived from four European countries was examined for plasmid profile, and outer membrane protein profile, by random amplified polymorphic DNA-PCR (RAPD-PCR) method, and digestions with restriction endonucleases were performed. All strains contained two large plasmids of approximately 60 and 70 kilobase pairs (kb). In addition, four strains contained a small plasmid of about 6 kb. These four strains produced cell-bound haemolysin only. Outer membrane protein analysis revealed subtle differences between strains. RAPD-PCR with primer I (CCGCAGCCAA) revealed 13 types, whereas primer II (AACGCGCAAC) yielded only two main types of different patterns. Results with primer I suggests a DNA sequence diversity within this species. The RAPD-PCR method provides a fast, economical and reproducible means for the typing of P. penneri. Digestion with restriction endonucleases indicated a high level of DNA methylation in this species.

Re-evaluation of in vitro activity of primycin against prevalent multiresistant bacteria

With the increasing emergence of antibiotic resistances old antibiotics became a valuable source to find agents suitable to address this problem. More than 20 years after the last report, our purpose was to re-evaluate the in vitro antibacterial activity of the topical agent primycin against current important bacterial pathogens. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of primycin were tested in comparison with agents widely applied topically, and with those of mupirocin and vancomycin, the topical and the non-topical gold-standard anti-MRSA agents. Primycin was ineffective (MIC>64 μg/ml) against all the Gram-negative isolates tested. On the other hand, all the tested Gram-positive isolates were susceptible with MIC90 values of 0.06 μg/ml for staphylococci and 0.5-1 μg/ml for enterococci, streptococci, and P. acnes isolates, including all the multiresistant strains. Against MRSA isolates primycin showed slightly higher activity than mupirocin, and inhibited the mupirocin-resistant strains also. MBC90 values ranged from 0.25 to 2 μg/ml for the investigated Gram-positive species. The bactericidal effect proved to be concentration-dependent in time-kill experiments. Spontaneous resistant mutants did not emerge in single-step mutation experiments and the resistance development was very slow by serial passaging. Passaged S. aureus strains showing increased primycin MIC values exhibited elevated vancomycin and daptomycin MIC values also. Though elucidation of the mechanisms behind warrants further investigations, these correlations can be related to development of vancomycin-intermediate phenotype. From the point of view of medical practice it is noteworthy that the increased primycin MIC values remained far below the concentration accessible by local application of the agent. These data make primycin a remarkable object of further investigations as well as a promising candidate for topical application against multiresistant Gram-positive pathogens.

Whole-Genome Draft Sequences of Nine Asymptomatic Escherichia coli Bacteriuria Isolates from Diabetic Patients

ABSTRACT Escherichia coli can colonize the urinary bladder without causing a disease response in the host. This asymptomatic bacteriuria (ABU) can protect against recurrent symptomatic urinary tract infection by virulent bacteria. Here, we report the whole-genome sequences of nine E. coli ABU isolates from diabetic patients.

Characterization of Asymptomatic Bacteriuria Escherichia coli Isolates in Search of Alternative Strains for Efficient Bacterial Interference against Uropathogens

Asymptomatic bacterial colonization of the urinary bladder (asymptomatic bacteriuria, ABU) can prevent bladder colonization by uropathogens and thus symptomatic urinary tract infection (UTI). Deliberate bladder colonization with Escherichia coli ABU isolate 83972 has been shown to outcompete uropathogens and prevent symptomatic UTI by bacterial interference. Many ABU isolates evolved from uropathogenic ancestors and, although attenuated, may still be able to express virulence-associated factors. Our aim was to screen for efficient and safe candidate strains that could be used as alternatives to E. coli 83972 for preventive and therapeutic bladder colonization. To identify ABU E. coli strains with minimal virulence potential but maximal interference efficiency, we compared nine ABU isolates from diabetic patients regarding their virulence- and fitness-associated phenotypes in vitro, their virulence in a murine model of sepsis and their genome content. We identified strains in competitive growth experiments, which successfully interfere with colonization of ABU isolate 83972 or uropathogenic E. coli strain 536. Six isolates were able to outcompete E. coli 83972 and two of them also outcompeted UPEC 536 during growth in urine. Superior competitiveness was not simply a result of better growth abilities in urine, but seems also to involve expression of antagonistic factors. Competitiveness in urine did not correlate with the prevalence of determinants coding for adhesins, iron uptake, toxins, and antagonistic factors. Three ABU strains (isolates 61, 106, and 123) with superior competitiveness relative to ABU model strain 83972 display low in vivo virulence in a murine sepsis model, and susceptibility to antibiotics. They belong to different phylogroups and differ in the presence of ExPEC virulence- and fitness-associated genes. Importantly, they all lack marked cytotoxic activity and exhibit a high LD50 value in the sepsis model. These strains represent promising candidates for a more detailed assessment of relevant fitness traits in urine and their suitability for therapeutic bladder colonization.

A modified bioautographic method for antibacterial component screening against anaerobic and microaerophilic bacteria

Direct bioautography is a useful method to identify antimicrobial compounds with potential therapeutic importance. Because of technical limitations till now, it has been applied only for aerobic bacteria. In this work we present the modification of the original method by which antimicrobial screening of bacteria requiring modified atmosphere became feasible by direct bioautography. Here we demonstrate its applicability by testing three anaerobic Clostridium perfringens and three microaerophilic Campylobacter jejuni strains against two essential oils, clove and thyme. Antimicrobial component profiles of clove and thyme essential oils against these two medically important pathogenic bacteria were compared and significant differences were revealed in their inhibition capacities. Linalool, a component of thyme essential oil exerted a more expressed antibacterial activity against C. perfringens than against C. jejuni. Our results demonstrate that direct bioautography is not only suitable for testing aerobic bacteria, but by applying the presently described modified version it can also contribute to the quest to find novel antimicrobial agents against multidrug resistant anaerobic and microaerophilic bacteria.