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.

Expression of the recombinant plasminogen activator (reteplase) by a non-lytic insect cell expression system.

Reteplase is a potent thrombolytic agent which is widely used in the management of acute myocardial infarction and stroke. It belongs to the third generation of the thrombolytic drugs and has been derived from native human tissue plasminogen activator by removing three domains of it and keeping the Kringle 2 and Serine protease domains. However, the high cost of this drug, has limited the application of this drug especially in the developing and third world countries. The most laborious steps in the bacterial production of this drug is its purification and refolding steps which keep the process yield low and the cost high. Therefore, in the present study we evaluated the expression of reteplase by a non-lytic insect cell expression system. Following cloning and transfection procedures, recombinant Sf9 insect cell clones expressing the reteplase protein were selected. Primarily, the expression was verified by dot-blot analysis and subsequently it was confirmed by Western Blotting showing a band of about 45 kD on nitrocellulose membrane. The biological activity of the expressed protein was also evaluated and showed to be about 29 IU/ml. This confirmed the possibility of expression and the correct folding of the expressed protein. Hence, optimization of the expression followed by purification of the protein could be the next steps of the study.

Assessment of selective toxicity of insect cell expressed recombinant A1-GMCSF protein toward GMCSF receptor bearing tumor cells.

One of the emerging therapeutic strategies for targeted treatment of most cancers is the use of immunotoxins which are fusion proteins consisted of a targeting and a toxic moieties. We previously showed that the recombinant A254-GMCSF fusion protein selectively kills acute myeloblastic leukemia cells which harbor a large number of granulocyte-macrophage colony stimulating factor (GMCSF) receptors. Since further in vitro and preclinical studies require large amounts of this fusion protein free from any troublesome material like lipopolysacharide, we selected the insect cell expression system. Thus, the coding sequences of the A254-GMCSF and its truncated form, A247-GMCSF, were cloned and expressed by Sf9 cells. Subsequently, specific cytotoxicity of the purified proteins was evaluated on GMCSF receptor positive cell lines. SDS-PAGE and Western blot analysis of the expressed A254GMCSF and A247GMCSF fragments revealed bands of about 60 kD which were larger than the theoretically predicted size of about 47 kD. Deglycosylation analysis showed that these proteins are N-glycosylated by the insect cells. However, any other post-translation modification of the proteins by insect cells could be the reason for higher molecular weight of the fragments. Cytotoxicity assays showed specific killing activity of these proteins on HL60 and U937 cell lines with IC(50)s ranging 2-2.5 µg/ml. These IC(50) values are much higher than those obtained from bacterially expressed A254-GMCSF (80 ng/ml) which could be due to any modification performed by insect cells on the fusion proteins.

Escherichia coli: a brief review of diarrheagenic pathotypes and their role in diarrheal diseases in Iran.

Diarrheagenic Escherichia coli have developed different strategies for establishment of infection in their host. Understanding these pathogenic mechanisms has led to the development of specific diagnostic tools for identification and categorization of E. coli strains into different pathotypes. This review aims to provide an overview of the various categories of diarrheagenic Escherichia coli and the data obtained in Iran pertaining to these pathotypes.

Cloning of fimH and fliC and expression of the fusion protein FimH/FliC from Uropathogenic Escherichia coli (UPEC) isolated in Iran.

BACKGROUND AND OBJECTIVES: Urinary tract infection (UTI) is one of the most common infections in the world. The majority of UTIs are caused by Uropathogenic Escherichia coli (UPEC) strains. FimH and FliC are the most important virulence factors of UPEC. To date, any ideal vaccine against UTI has not been approved for human use and we need to test new targets to develop an ideal vaccine against UTI. In this study, we constructed fusion fimH/fliC of UPEC as a novel vaccine candidate against UTI. MATERIAL AND METHODS: PCR amplification of fimH and fliC genes of the UPEC isolates was performed by specific primers designed for this purpose. Construction of fimH/fliC hybrid gene was performed by overlap PCR. The fimH, fliC and fimH/fliC were cloned in pET28a vector. The confirmation of expression of the proteins was done by SDS-PAGE and Western blot. RESULTS: The fliC and fimH genes were amplified in all of the UPEC isolates tested. The fimH showed significant homology with the sequences in GenBank. We generated a fusion consisting of the fimH linked to the N-terminal end of fliC. Sequencing of the fusion fimH/fliC showed that fusion was constructed correctly. SDS-PAGE and western blot confirmed the expression of the proteins in optimized condition. CONCLUSION: Urinary tract infection is a huge burden on healthcare system in many countries. UPEC is isolated in around 80% of UTI cases. Antibiotic therapy resulted in the emergence of antibiotic resistance in UPEC strains. This is the major cause for an increasing requirement for a vaccine to prevent UTI. This work describes for the first time the construction of a novel fusion protein from Iranian UPEC isolates. Further immunological studies are required for evaluation of this protein as a novel and safe vaccine candidate against UTI caused by UPEC.

Phenotypic and Genotypic Characterization of Enteropathogenic Escherichia coli (EPEC) strains in Tehran, Iran.

BACKGROUND AND OBJECTIVES: Enteropathogenic Escherichia coli (EPEC) strains can be detected by serogrouping and the presence of enterocyte attaching- effacing (eae) gene. Most EPEC strains belong to a certain O antigenic group. Locus of enterocyte effacement (LEE) Pathogenicity Island contains the eae gene and secretory proteins (ESPs) that introduce the attaching-effacing lesion. LEE inserted in tRNA genes include the SelC, PheU and PheV sites. The aim of the present study was to genetically characterize EPEC strains isolated from children with diarrhea. MATERIALS AND METHODS: Serogrouping was performed by EPEC antisera in 321 E. coli isolates. The presence of eae, stx, espB, and eaf genes and detection of insertion sites of LEE was done by PCR using specific primers. RESULTS: Seventeen (5.3%) isolates belonging to 7 EPEC serogroups were identified among the whole material and all carried the eae gene. None of the 321 isolates showed presence of stx gene indicating that all 17 isolates classified as EPEC by O serogrouping did not belong to the enterohaemorrhagic E. coli (EHEC) group. Of these, 8 (53%) isolates carried the eaf and 16 (94.1%) carried the espB gene. The insertion sites of LEE in serogrouped isolates were selC (in 6 isolates), pheU (in 7 isolates) and pheV (in 2 isolates). The insertion site in 2 isolates was not determined by PCR. CONCLUSION: Serogrouping and detection of the eae gene showed to be reliable for detection of EPEC strains. No Shigatoxin- producing E. coli (STEC) strain was found among the isolates. Detection of the insertion site of LEE showed that selC, pheU or PheV are insertion sites of LEE in the EPEC strains.

A recombinant hybrid peptide composed of AAF adhesin of enteroaggregative Escherichia coli and Shiga toxin B subunit elicits protective immune response in mice.

Shiga toxin producing Escherichia coli (STEC) are a group of diarrheagenic Escherichia coli (E. coli) whereby Shiga toxin is the main virulence factor. It is composed of an A subunit, which mediates toxicity, and a B subunit (StxB), which is a nontoxic homopentameric protein responsible for toxin binding and internalization into target cells by interacting with the glycolipid, globotriaosylceramide (Gb3). Enteroaggregative Escherichia coli (EAEC) are a group of E. coli with aggregative adherence to epithelial cells, which play an important role in its pathogenesis. EAEC are the cause of diarrhea in developing countries and in the developed world. Aggregative adherence fimbria (AAF) of EAEC represents the adhesin that confers the presence of aggregative adherence (AA) phenotype on EAEC strains. The gene encoding non-toxic B subunit of Shiga toxin (StxB) was coupled to aggregative adherence fimbriae (AAF) of the EAEC structural gene. The resulting polypeptides (B-AAF/I, B-AAF/II) were designed to elicit immune response in immunized mice with recombinant peptides. The antibody, hence obtained, inhibited the adherence of prototype EAEC strains to HeLa cells and, on the other hand, protected the immunized mice against a lethal dose of Shiga toxin. Therefore, this promising data could indicate that this kind of polypeptide strategy is a good candidate for any probable vaccine design against diarrheal infection.

Distribution of genes encoding toxins and antibiotic resistance patterns in diarrhoeagenic Escherichia coli isolates in Tehran.

Pathogenicity of Escherichia coli can involve a large number of virulence factors, toxins being the most obvious. We assessed the distribution of genes encoding toxins among E. coli isolates from diarrhoeal cases using DNA probes. From 200 isolates, 92 (46.0%) carried genes encoding for toxins, 43.5% of these being multitoxigenic. Enteroaggregative heat-stable enterotoxin was detected in 40 (43.5%) isolates, verotoxin in 38 (41.3%), cytolethal distending toxin in 24 (26.1%), heat-stable enterotoxin in 12 (13.0%) and heat-labile enterotoxin in 10 (10.9%). Furthermore, 40 strains (70.0%) carried resistance. We conclude that toxigenicity and antibiotic resistance are the main contributing factors leading to the virulence potential of these E. coil isolates.

Distribution of genes encoding toxins and antibiotic resistance patterns in diarrhoeagenic Escherichia coli isolates in Tehran

Pathogenicity of Escherichia coli can involve a large number of virulence factors, toxins being the most obvious. We assessed the distribution of genes encoding toxins among E. coli isolates from diarrhoeal cases using DNA probes. From 200 isolates, 92 [46.0%] carried genes encoding for toxins, 43.5% of these being multitoxigenic. Enteroaggregative heat- stable enterotoxin was detected in 40 [43.5%] isolates, verotoxin in 38 [41.3%], cytolethal distending toxin in 24 [26.1%], heat- stable enterotoxin in 12 [13.0%] and heat- labile enterotoxin in 10 [10.9%]. Furthermore, 40 strains [70.0%] carried resistance. We conclude that toxigenicity and antibiotic resistance are the main contributing factors leading to the virulence potential of these E. coli isolates

Selective cytotoxicity of recombinant STXA1-GM-CSF protein in hematopoetic cancer cells.

Chimeric proteins are composed of a cell-targeting moiety and a cell-killing moiety. In this study, a chimeric protein, STXA1-GM-CSF, composed of catalytic domain of Shiga toxin (A1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed and expressed in E. coli. Cytotoxicity, receptor blocking, and neutralization experiments revealed that the chimeric protein induced cytotoxic effect on different cell lines. This effect was found to be specific, due to the presence of the killing moiety (A1), which exerts its effect through a specific GM-CSF-targeting domain, by binding to its receptor present on those cell lines. These initial investigations indicate that the chimeric protein was functional; further analyses are required for its application.

Comparison of polymerase chain reaction systems for detection of different cdt genes in Escherichia coli strains.

AIMS: Cytolethal distending toxins (CDT) are tripartite toxins encoded by three adjacent or overlapping genes (cdtA, cdtB, cdtC) and found in multiple pathogens. The present knowledge regarding heterogeneity of cdt genes and our previous study revealed that the available polymerase chain reaction (PCR) systems lack adequate specificity. The detection of various cdt genes present in Escherichia coli strains, from different geographical regions demands further assays for wide-range coverage. On the basis of these observations, we were prompted to undertake the present study; hence the specificity of existing PCR systems was addressed using E. coli prototype strains with known cdt gene sequences. METHODS AND RESULTS: A multiplex PCR designed for the detection of E. coli cdt genes was found to be sensitive and specific enough for initial screening. However, for subtyping, the PCR systems yielded nonspecific products upon amplification. These primers are usually designed for sequences of the cdtB locus (the most conserved region of the gene), and since CDT-producing E. coli strains carry different cdt genes, none of the systems are really type specific. Furthermore, PCR systems with type-specific primers for other regions of the gene, i.e. ORF A or ORF C are found to be strain specific and their applications in different geographical regions have limitations. CONCLUSIONS: In conclusion, based on our observations, using these available primers, it seems that the existing PCR systems are not sufficiently accurate to differentiate between different types of cdt genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained from this study revealed that so-far reported PCR systems are short in specificity. These PCR protocols were not found to be specific enough to detect various cdt genes and have a limited range of application. Moreover, due to similarities in cdt genes the cross-reaction between different sets of primers exists. Hence for epidemiological studies, some additional PCR protocols are required for screening clinical isolates for cdt genes.

Distribution of virulence related genes among enteroaggregative Escherichia coli isolates: using multiplex PCR and hybridization.

The importance of enteroaggregative Escherichia coli (EAEC) strains in public health around the world is becoming increasingly clear. EAEC diagnosis has long been problematic. In this study, the recently designed multiplex PCR based on three plasmid-borne genes (AA probe, aap, and aggR) and DNA hybridization assay with plasmid-derived DNA probes were used for detection of HEp-2 adherent strains. These were isolated from an epidemiologic study of diarrhea in Iran. Using AA and DA probes revealed that 32.4%, 16.2%, 23%, and 28.4% of these isolates were AA, DA, AA/DA, and non-AA/DA, respectively. However, employing multiplex PCR for detection of these isolates, showed that 51.3% of the strains were AA and the rest 48.7% were not AA. While presence of other genes (pet, shf, aggA, aafA) considered to be specific for EAEC were checked among these isolates. The data obtained revealed that except for AA, aap, and aggR, the rest of the virulence related genes are not specific for EAEC isolates and are randomly distributed among adherent isolates. Over all the results obtained here indicated that this multiplex PCR is specific and sensitive assay. Phenotypically adherent strains are divided into two main groups, by use of this multiplex PCR i.e., typical EAEC isolates that carry the three plasmid-borne genes all together and atypical EAEC isolates in which the three genes are not linked together.

Short report: characterization of enteroaggregative Escherichia coli isolates from Iranian children.

We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Iranian infants and children. To better understand the characteristics of EAEC in Iran, we analyzed EAEC isolates for the presence of pAA plasmid-borne factors. Ninety-eight E. coli strains that displayed the aggregative adherence (AA) pattern on HeLa cells were hybridized with the CVD432 (AA) probe and with genes encoding enteroaggregative heat-stable enterotoxin-1 and aggregative adherence fimbriae (AAF) I and II. Our data suggest that AAF/II is common in this population and that AAF/I and AAF/II can sometimes be detected in the same E. coli isolate. Surprisingly, we have found that AA probe-negative strains in Iran share virulence factors with AA probe-positive isolates and therefore may be more similar to probe-positive strains than previously believed.

Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048-5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr(+) adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr(-) adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr(-) adhesin) was found to predominate in afa-positive isolates from sepsis patients (75%); it was frequent in afa-positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr(+) strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa-positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by afa-positive strains.

Detection and characterization of verotoxin-producing strains among sorbitol non-fermenting Escherichia coli.

The presence of genes for verotoxin 1 and 2 (VT1 and 2) among sorbitol non-fermenting Escherichia coli isolates from diarrhoeal cases was assessed using polymerase chain reaction assay. Of 60 (88%) positive isolates, 37 (62%) harboured VT1 and 23 (38%) both VT1 and VT2. In HeLa cell adherence assay, 48 (71%) isolates exhibited mannose-resistant adherence to HeLa cells. Multidrug resistance was observed in 56 (82%) isolates, with ampicillin, chloramphenicol, streptomycin, sulfamethoxazole-trimethoprim and tetracycline pattern being the most common. There were 13 common and 22 single biochemical phenotypes identified. Isolates belonging to common biochemical phenotypes normally had a similar pattern of adherence and VT production, but differed greatly in their pattern of antibiotic resistance, pointing to a high rate of antibiotic-resistance transfer among these isolates.

Detection and characterization of verotoxin-producing strains among sorbitol non-fermenting Escherichia coli

The presence of genes for verotoxin 1 and 2 [VT1 and 2] among sorbitol non-fermenting Escherichia coli isolates from diarrhoeal cases was assessed using polymerase chain reaction assay. Of 60 [88%] positive isolates, 37 [62%] harboured VT1 and 23 [38%] both VT1 and VT2. In HeLa cell adherence assay, 48 [71%] isolates exhibited mannose-resistant adherence to HeLa cells. Multidrug resistance was observed in 56 [82%] isolates, with ampicillin, chloramphenicol, streptomycin, sulfamethoxazole-trimethoprim and tetracycline pattern being the most common. There were 13 common and 22 single biochemical phenotypes identified. Isolates belonging to common biochemical phenotypes normally had a similar pattern of adherence and VT production, but differed greatly in their pattern of antibiotic resistance, pointing to a high rate of antibiotic-resistance transfer among these isolates

Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli.

The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E. coli (EPEC) by slide agglutination was evaluated using hybridization and PCR. The adherence property of these strains was assayed using 3h HeLa cells adherence assay. The results obtained indicated that virulence-associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes. In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent. Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P

Verotoxin-producing Escherichia coli (VTEC) infection in randomly selected population of Ilam Province (Iran).

In a randomly selected population, 2,008 fecal samples were screened for presence of Verotoxin producing Escherichia coli (VTEC) by colony sweep polymyxin-B extraction method. Non-sorbitol fermentation (NSF) phenotype and slide agglutination with O157: H7 antisera were used for screening and detection of this serotype. Ninety-eight (4.9%) fecal samples were found to be VTEC-positives and none of them belonged to the O157: H7 serotype. In rural areas, most individuals carrying VTEC isolates were asymptomatic, whereas in urban areas, a significant association was found between VTEC isolation and diarrhoea (p < 0.01).

Adherence of non-enteropathogenic Escherichia coli to HeLa cells.

Three hundred and nine strains of Escherichia coli isolated from infants and children with diarrhoea but not belonging to any recognised classes of diarrhoeagenic E. coli were investigated for their ability to adhere to HeLa cells in the presence of D-mannose. An enteroadherent-aggregative pattern (EAgg) was observed in 32.03%, localised adherence (LA) in 4.5%, diffuse adherence (DA) in 5.8%, and LA/DA and EAgg/LA in 1.9% and 1.2% of the isolates respectively. The results obtained with 100 control isolates were: EAgg 17%, LA 2%, DA 2%, LA/DA 2%, EAgg/LA 6% and DA/EAgg 1%. No adherence was manifested by 168 (54.36%) of 309 diarrhoeal isolates and 70% of the 100 control isolates. The results of this study showed that amongst non-enteropathogenic E. coli, strains exhibiting the EAgg pattern are significantly associated with diarrhoea (p < 0.005). Most of these strains showed a pattern of multiple drug resistance.