The genotypic and phenotypic characteristics contributing to high virulence and antibiotics resistance in Escherichia coli O25-B2-ST131 in comparison to non- O25-B2-ST131.

BACKGROUND: Escherichia coli serogroup O25b-sequence type 131 (E. coli O25-B2-ST131) is considered as multidrug-resistant and hypervirulent organism. There is lack of data about involvement of this pathogen in the children's infection. In this study, the prevalence, and clonality, virulence capacity, and antibiotic resistance phenotype and genotype of E. coli O25-B2-ST131 compared with non-O25-B2-ST131 isolates were investigated in children with urinary tract infection in Tehran, Iran. METHODS: The E. coli isolates from urine samples were identified using conventional microbiological methods. Characterization of E. coli O25-B2-ST131 clone, antibiotic susceptibility, biofilm formation, ESBLs phenotype and genotype, serum resistance, hemolysis, hydrophobicity, and formation of curli fimbriae were done using conventional microbiological and molecular methods. Clonality of the isolates was done by rep-PCR typing. RESULTS: Among 120 E. coli isolates, the highest and lowest antibiotic resistance was detected against ampicillin (92, 76.6%) and imipenem 5, (4.1%), respectively. Sixty-eight (56.6%) isolates were ESBL-producing and 58 (48.3%) isolates were considered as multi-drug resistance (MDR). The prevalence of ESBL-producing and MDR isolates in O25-B2-ST131 strains was higher compared with the non-O25-B2-ST131 strains (p value < 0.05). O25-B2-ST131 strains showed significant correlation with serum resistance and biofilm formation. Amongst the resistance and virulence genes, the prevalence of iucD, kpsMTII, cnf1, vat, blaCTX-M-15, and blaSHV were significantly higher among O25-B2-ST131 isolates in comparison with non-O25-B2-ST131 isolates (p value < 0.05). Considering a ≥ 80% homology cut-off, fifteen different clusters of the isolates were shown with the same rep-PCR pattern. CONCLUSIONS: Our results confirmed the involvement of MDR-ESBLs producing E. coli strain O25-B2-ST131 in the occurrence of UTIs among children. Source tracking and control measures seem to be necessary for containment of the spread of hypervirulent and resistance variants in children.

In silico design and in vitro validation of a novel PCR-RFLP assay for determination of phylogenetic clusters of streptokinase gene alleles in streptococci groups.

Streptokinase (SK), a heterogeneous plasminogen (Pg) activator protein secreted by groups A, C and G streptococci (GAS/GCS/GGS) is a virulence factor composed of three structural domains; SKα/SKß/SKγ. Phylogenetic analysis of the major variable region of SKß (sk-V1; nucleotides 448-791; 343bp) which classifies the SK alleles into SK1/SK2 clusters and SK2a/SK2b sub-clusters, is an approved assay to categorize clinical/natural streptococcal-isolates into co-related functional/pathogenesis groups. Herein, we describe a novel PCR-RFLP assay that in combination with Numerical Taxonomy and multivariate analysis System (NTSYS) resulted to dendrograms with complete adaption to that of the phylogenetic analysis of sk-V1-based clustering. In silico analyses by 30 restriction enzymes on GenBank-acquired sk-V1 sequences of known streptococcal clusters, resulted to the selection of "BsrI, MseI and Tsp45I″ enzymes that produced proper patterns to construct the expected dendrograms. In vitro analysis of the selected enzymes on clinical isolates of GAS/GCS/GGS validated the production of the same in silico-observed digestion patterns. Comparison of the constructed dendrogram and phylogenetic trees of selected GenBank and clinical isolates of streptococci indicated complete adaptation. Assessment of Pg-activation activity in selected clinical isolates indicated the expected co-related functionalities of the classified SK-clusters by the invented PCR-RFLP/NTSYS method. The simplicity of the assay relieves the need of sequencing/phylogenetic analyses for SK-clustering.

Different Virulence Capabilities and ompA Expressions in ST2 and ST513 of Multidrug-Resistant Acinetobacter baumannii.

Successful clones of Acinetobacter baumannii cause a variety of nosocomial infections through serum resistance, biofilm formation, and antimicrobial resistance as virulence capabilities. Fifty clinical isolates of multidrug-resistant (MDR) A. baumannii were analyzed for clonal relatedness, serum resistance, biofilm formation, and in vivo assays. Furthermore, some virulence genes, sequence variation of ompA, and its expression were studied. The MLST (multilocus sequence typing) results showed that there were three sequence types among MDR isolates including ST2 (64%, 32/50), ST513 (30%, 15/50), and ST1 (6%, 3/50). The data showed that the clinical isolates recovered from sputum had mostly high biofilm-formation capacity, while isolates recovered from host interior fluids had high serum resistance. The results of PCR assays and in silico analysis represented patterns of virulence genes and even ompA sequence variations among MDR isolates which were clonally dependent. While quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that bacteremia-producing strains in C57/BL6 mice significantly overexpress ompA (P < 0.05) and have a direct relation with the level of IL-6 in bloodstream of mice. Moreover, the expressions of ompA among indistinguishable clones (ST2 or ST513) were clonally independent.

Sequence and kinetic analyses of streptokinase from two group G streptococci with high fibrin-dependent plasminogen activities and the identification of novel altered amino acids as potential hot spots.

OBJECTIVE: To gain insights on the degree of heterogeneity and kinetic differences of streptokinase (SK) from group G (SKG) Streptococci compared with standard SK from group C (SKC) and identification of potentially contributing critical residues (hotspots). RESULTS: DNA and sequencing analyses confirmed the proper construction of all SK encoding vectors (two SKGs and one standard SKC). SDS-PAGE and western blot analyses confirmed the expression and proper purification of the recombinant SKs from E.coli with the expected size of 47 kDa. Kinetic analyses of two SKGs, compared with SKC, showed higher levels of specific [(×103 IU/mg) of 725 and 715 vs. 536] and fibrin-dependent proteolytic activities [Kcat/KM (min-1/µM) of 37 and 30 vs. 23], accompanied by declined fibrin-independent amidolytic activities [Kcat/KM (min-1/mM) of 109 and 84 vs. 113], respectively. Sequence alignments identified 10 novel residual substitutions scattered in SKα (I33F, R45Q, SKG132, A47D, and G55 N), SKß (N228 K, F287I), and SKγ domains (L335 V, V396A, T403S) of SKGs, as potential hotspots. CONCLUSION: The residue substitutions identified might critically contribute as hot spots to different kinetic parameters of SKGs and might assist in further elucidation of structure/function relations and rational design of SKs with improved (fibrin-dependent) therapeutic properties.

Tagging staphylococcal enterotoxin B (SEB) with TGFaL3 for breast cancer therapy.

Recent research has attempted to direct superantigens towards tumors by means of tumor-targeted superantigen (TTS) strategy. In this study, we explored the antitumor property of TTS by fusing the third loop of transforming growth factor α (TGFαL3) to staphylococcal enterotoxin type B (SEB) and investigated the possibility of the therapeutic application of TGFαL3-SEB as a novel antitumor candidate in mice bearing breast cancer. Treatment was performed through intratumoral and intravenous injection of TGFαL3-SEB. Tumor size/volume, long-term survival, and cytokine secretion were assessed. In addition, the toxicity of each treatment on liver and kidneys was examined. Our results indicated that the relative tumor volume significantly increased in the mice receiving intratumoral TGFaL3-SEB (p < 0.05). Surprisingly, 5 out of the 14 mice were cleared from the tumor thoroughly in 10-25 days after intratumoral administration of TGFaL3-SEB. Quantification of cytokines clearly showed that the mice receiving intratumoral SEB significantly secreted higher interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) compared with the other groups (p < 0.05). The antitumor effect was followed by inhibition of cell proliferation (Ki-67) and micro vascularization (CD31). The highest and lowest levels of tumor necrosis were observed in the intratumoral administration of TGFαL3-SEB (85 %) and PBS (14 %), respectively. Intratumoral injection of TGFαL3-SEB increased the lifespan of the mice so 37.5 % of them could survive for more than 6 months (p < 0.05). Overall, our findings indicated that intratumoral administration of TGFαL3-SEB effectively inhibited the growth of breast tumors through induction of necrosis and suppressing proliferation and angiogenesis without systemic toxicity.

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK.

Streptokinase (SK), the heterogeneous protein family secreted by some groups of ß-hemolytic streptococci (ßHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 ßHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI-NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10(4) IU mg(-1)). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.

Design of a biological method for rapid detection of presence of PCR inhibitors in aged bone DNA.

BACKGROUND: Molecular human identification is one of the most important tests performed in forensic laboratories. Some of these tests are applied for identification of human remains from natural disasters, wars, etc., but problems may occur as a result of DNA degradation and external DNA contamination. We investigated effects of bacterial DNA on identifying the presence or absence of PCR inhibitors in aged bone DNA. METHODS: DNA samples were extracted from blood, bone remains and Escherichia coli. These DNA were amplified using human and bacterial specific primers. RESULTS: Using different blood, aged bone, and bacterial DNA dilutions along with PCR based methods; we checked their positive, negative effects, or detecting presence of inhibitors in aged bone DNA by PCR method. CONCLUSIONS: Our observation indicated that the addition of bacterial DNA could be a valid biological method for testing the quality of bone DNA to enable us to obtain a usable profile for the identification of human remains. This method will help to test the presence of inhibitors, quantity or even quality of DNA which are of importance in profiling archeological remains. Our method will help to determine if PCR failure is due to presence of inhibitors or lack of amplifiable DNA either because of degradation, minute amount or absence of human DNA.

Prevalence of O25b-ST131 Escherichia coli Clone: Fecal Carriage of Extended-Spectrum ß-Lactamase and Carbapenemase-Producing Isolates in Healthy Adults in Tehran, Iran.

Fecal carriage of multidrug-resistant Escherichia coli, particularly sequence type 131 (ST131), is becoming a global concern. This study aimed at determining the prevalence rate and molecular epidemiology of extended-spectrum ß-lactamase-producing E. coli (ESBL-Ec), carbapenemase-producing E. coli (CPEc), ceftazidime/avibactam (CAZ/AVI)-resistant E. coli, and ST131 isolates in healthy fecal carriers in Tehran, Iran. Among 540 samples studied, 233 (43.1%) carried ESBL-Ec, with the majority (93.9%) harboring the blaCTX-M. The carriage rate of CPEc was 2.5% (n = 14/540), and blaNDM gene was the predominant carbapenemase gene. Most CPEc isolates (n = 11/14) was shown to be resistant to CAZ/AVI. Among ESBL-Ec/CPEc, 7.3% (n = 17/233) belonged to E. coli ST131 clone, which was identified by polymerase chain reaction and confirmed by multilocus sequence typing. The ST131 isolates genetically typed by pulsed-field gel electrophoresis were heterogeneous and four different plasmids were detected by plasmid typing, with the IncFIA/FIB being the major type. Our findings disclose that the presence of carbapenem-resistant ST131 isolates, which are also resistant to CAZ/AVI, contributes to the spread of resistant strains in the community. Therefore, screening and monitoring of such resistant clone in healthy people is necessary.

Distribution of ciprofloxacin-resistance genes among ST131 and non-ST131 clones of Escherichia coli isolates with ESBL phenotypes isolated from women with urinary tract infection.

BACKGROUND AND OBJECTIVES: Escherichia coli (E. coli) sequence type 131 (ST131) is associated with extended-spectrum beta-lactamase (ESBL) production and fluoroquinolone resistance. This study aimed to investigate the prevalence of ST131, ESBL, and plasmid-mediated quinolone resistance (PMQR) genes in the ciprofloxacin-resistant (CIPR) and ESBL producers from women with UTI. MATERIALS AND METHODS: The CIP-resistant ESBL producing (CIPR/ESBL+) E. coli isolates were screened for ST131-by specific PCR of mdh and gyrB. The ESBL and PMQR genes were screened by single PCR. The ST131 and non-ST131 isolates were selected to determine the mutations of gyrA and parC using PCR and sequencing, and also their genetic background by the Pasteur-MLST scheme. RESULTS: Overall, 55% (33/60) CIPR/ESBL+ isolates were identified as ST131 (94% O25b-ST131). Resistance rate to ampicillin-sulbactam (70%), aztreonam (97%) and gentamicin (61%), the prevalence of aac(6')-Ib-cr (66%), bla CTX-M-15 (82%), the profile of qnrS+aac(6')-Ib-cr (30%), and the double mutation in the parC was significantly higher in ST131 than non- ST131 isolates. The coexistence of PMQR and ESBL genes was found in more than 50% of ST131 and non-ST131 isolates. ST131 isolates differentiated into PST43 and PST506. CONCLUSION: Management of women with UTI caused by the CIPR/ESBL+ isolates (ST131) co-harbored PMQR, ESBL, and chromosomal mutations, is important for their effective therapy.

Distribution of Pathogenicity Island Markers and H-Antigen Types of Escherichia coli O25b/ST131 Isolates from Patients with Urinary Tract Infection in Iran.

Escherichia coli serogroup O25b-sequence type 131 (E. coli O25b/ST131) is known as a multidrug-resistant organism with high virulence potential and has received attention internationally. We aim to investigate the prevalence of O25b/ST131 and the distribution of blaCTX-M-15, pathogenicity island (PAI) markers, phylogenetic groups, and H-antigen typing in the E. coli O25b/ST131 isolated from patients with urinary tract infection (UTI) in Tehran, the capital of Iran. Seventy (26.9%) E. coli isolates were identified as O25b/ST131. There was also a significant difference in the prevalence of virulence genes, including papA, sfa, sat, cnf1, iutA, kpMII, traT, and usp, in the O25b/ST131 isolates rather than non-O25b/ST131 ones (p ≤ 0.05). Furthermore, 78% of the O25b/ST131 isolates carried four to seven PAIs, while 71% of non-O25b/ST131 isolates carried two to four PAI markers (p ≤ 0.05). Our study showed that in addition to H4, other H-antigens may play a role in the O25b/ST131 virulence potential. Besides, a significant association was found between the history of previous UTIs and infection among the O25b/ST131 clone isolates. Pulsed-field gel electrophoresis revealed circulating of O25b:H4-ST131/PST43 clone in both hospital and community. Approximately one in every three uropathogenic E. coli isolates was the O25b/ST131 clone, representing a significant public health threat. Practical investigation on O25b/ST131 can be helpful in better understanding of ST131 evolution and controlling UTI in hospitals.

Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen activation: A Domain-Exchange Study

Background: Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKß, and SK). Based on the variable region of SKß, GAS-SK (ska) are clustered as SK1 and SK2 (including cluster2-streptokinase (SK2a)/SK2b), which show low and high fibrinogen (FG)-dependent plasminogen (Plg) activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (skcg) variants display properties similar to SK1. Herein, by SKß exchange between GGS (G88) and GAS-SK2a (STAB902) variants, the potential roles of SK domains in amidolytic/proteolytic activity and FG-bound-Plg activation are represented. Methods: Two parental SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKß-exchanged constructs (SKC1: αG88-ßSTAB-γG88 and SKC2; αSTAB-ßG88-γSTAB) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SK were expressed in E. coli and purified by nickel-nitriloacetic acid chromatography. PA potencies of SKs were measured by colorimetric assay. Results: SDS-PAGE and Western-blot analyses confirmed the proper expression of 47-kDa SK. Analyses indicated that the catalytic efficiency (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SKß, respectively. The increase of FG-bound-Plg activation for SKSTAB902/SKC1 containing SK2aß was around six times, whereas for SKG88/SKC2 containing skcgß, it was four times. Conclusion: Although SKß has noticeable contribution in FG-bound-Plg activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK.

Characterization of virulence genes of non-O157 Shiga toxin-producing Escherichia coli isolates from two provinces of Iran.

Shiga toxin-producing Escherichia coli (STEC) include O157:H7 and non-O157 serotypes. The public health impact of STEC infections is high because of their ability to cause severe infections. We characterized our STEC strains isolated from diarrheal and asymptomatic persons in northern and southwest Iran. The 29 STEC strains were examined for the presence of virulence genes by polymerase chain reaction (PCR) and their H type was analyzed by PCR-restriction fragment length polymorphism (RFLP) of the fliC gene. Moreover, the adherence properties of these strains were checked by HeLa cell adherence assay. The presence of non-O157 isolates under these conditions was again verified. The stx1 gene was present in 93% of the isolates, and the gene encoding intimin (eae) was not found to be present among the isolates. Almost all of the STEC isolates, with the exception of three, were non-adherent upon tissue culture assay. The serogrouping revealed the presence of seven different O types among non-O157 isolates. PCR-RFLP results for the fliC gene and classical serology examination with H antisera indicated the presence of nine different H types. In this study, three new serotypes of non-O157:H7 (i.e., O25:H3, O85:H32, and O162:H21) were found to be Shiga toxin producers. These findings reconfirm the results of our previously reported studies showing that non-O157:H7 serotypes are more prevalent under the present conditions. More detailed characterization of these isolates will require additional genetic studies.

The ß-domain of streptokinase affects several functionalities, including specific/proteolytic activity kinetics.

Streptokinase (SK) is a plasminogen activator which converts inactive plasminogen (Pg) to active plasmin (Pm), which cleaves fibrin clots. SK secreted by groups A, C, and G Streptococcus (SKA/SKC/SKG) is composed of three domains: SKα, SKß and SKγ. Previous domain-swapping studies between SK1/SK2b-cluster variants revealed that SKß plays a major role in the activation of human Pg. Here, we carried out domain-swapping between skcg-SK/SK2-cluster variants to determine the involvement of SKß in several SK functionalities, including specific/proteolytic activity kinetics, fibrinogen-bound Pg activation and α2 -antiplasmin resistance. Our results indicate that SKß has a minor to determining role in these diverse functionalities for skcg-SK and SK2b variants, which might potentially be accompanied by few critical residues acting as hot spots. Our findings enhance our understanding of the roles of SKß and hot spots in different functional characteristics of SK clusters and may aid in the engineering of fibrin-specific variants of SK for breaking down blood clots with potentially higher efficacy and safety.

Computational simulations assessment of mutations impact on streptokinase (SK) from a group G streptococci with enhanced activity - insights into the functional roles of structural dynamics flexibility of SK and stabilization of SK-µplasmin catalytic complex.

Streptokinase (SK), a plasminogen activator (PA) that converts inactive plasminogen (Pg) to plasmin (Pm), is a protein secreted by groups A, C, and G streptococci (GAS, GCS, and GGS, respectively), with high sequence divergence and functional heterogeneity. While roles of some residual changes in altered SK functionality are shown, the underlying structural mechanisms are less known. Herein, using computational approaches, we analyzed the conformational basis for the increased activity of SK from a GGS (SKG132) isolate with four natural residual substitutions (Ile33Phe, Arg45Gln, Asn228Lys, Phe287Ile) compared to the standard GCS (SKC). Using the crystal structure of SK.Pm catalytic complex as main template SKC.µPm catalytic complex was modeled through homology modeling process and validated by several online validation servers. Subsequently, SKG132.µPm structure was constructed by altering the corresponding residual substitutions. Results of three independent MD simulations showed increased RMSF values for SKG132.µPm, indicating the enhanced structural flexibility compared to SKC.µPm, specially in 170 and 250 loops and three regions: R1 (149-161), R2 (182-215) and R3 (224-229). In parallel, the average number of Hydrogen bonds in 170 loop, R2 and R3 (especially for Asn228Lys) of SKG132 compared to that of the SKC was decreased. Accordingly, residue interaction networks (RINs) analyses indicated that Asn228Lys might induce more level of structural flexibility by generation of free Lys256, while Phe287Ile and Ile33Phe enhanced the stabilization of the SKG132.µPm catalytic complex. These results denoted the potential role of the optimal dynamic state and stabilized catalytic complex for increased PA potencies of SK as a thrombolytic drug.

Epitope-based immunoinformatics study of a novel Hla-MntC-SACOL0723 fusion protein from Staphylococcus aureus: Induction of multi-pattern immune responses.

Staphylococcus aureus infections are now one of the most common causes of surgical drainage, bacteremia, and hospital-acquired infections. The emergence of antibiotic resistance has increased mortality and costs of treatment. The design of a new vaccine against S. aureus would have a great beneficial impact on public health. In the current report, we design and introduce a novel epitope-based fusion protein (Hla, MntC and SACOL0723) and investigate its biological activities. Three known antigenic proteins from S. aureus were analyzed for the prediction of immunogenic B and T-cell epitopes and validated using bioinformatics tools. The affinity and the map of interactions between the receptor and ligand were evaluated via docking protocols. Functional activity of the recombinant protein was assessed by western blot and opsonophagocytosis tests and determining the bacterial burden from the infected tissues. To determine the type of induced immunity, cytokines profile and isotyping ELISA was performed. Based on in silico analysis, seven epitopes rich domain including highly scored T and B-cell epitopes were selected. The study results indicated that the high titer of specific antibodies raised against the vaccine candidate could opsonize the bacteria and decrease the viable bacterial cells. The fusion protein was able to elicit a mixture of Th1, Th2, and Th17 immune responses more towards Th1 and Th17. In conclusion, the fusion protein formulated with alum could be considered as a potential vaccine candidate for protection against S. aureus in the near future.

Molecular analysis and genotyping of pathogenicity locus in Clostridioides difficile strains isolated from patients in Tehran hospitals during the years 2007-2010.

BACKGROUND & AIMS: Clostridioides difficile (C. difficile) has been identified as the leading cause of antibiotic associated diarrhea (AAD). Co-carriage of an intact pathogenicity locus (PaLoc) with binary toxin genes in C. difficile strains seems to be linked with severe disease outcomes in the infected patients. Epidemiology of C. difficile infection (CDI) in hospital setting and knowledge about their genetic context help us to decrease the morbidity, mortality, and costs associated with Clostridioides difficile infection. In the present study was aimed to characterize genetic diversity of PaLoc among different C. difficile strains isolated from hospitalized patients and carriage of cytolethal distending toxin gene (cdt) in different hospitals. METHOD: C. difficile strains were isolated from stool samples of inpatients referred to a reference laboratory from different hospitals and also outpatients with diarrhea, during 2008-2011. DNA was extracted from pure culture of the bacterium and PCR was performed for tcdA, tcdB, tcdE, tcdC, tcdD, and cdu2 genes. Carriage of two binary toxin genes cdtA, cdtB was also determined in these strains. To find clonal strains, similarity of genotypes and integrity of PaLoc among the isolates was compared in each hospital. RESULTS: The intact PaLoc was found most frequently among the isolates in the outpatients (19/51, 37.2%, Group I), while incomplete PaLoc found mostly in patients who were hospitalized in the infectious diseases and internal diagnosis wards. tcdA and tcdB genes were detected in different combinations among the studied strains. These strains showed tcdA+B+, tcdA+B-, and tcdA-B+ genotypes in a frequency of 76.4% (39/51), 7.8% (4/51), and 17.6% (9/51), respectively. Analysis of gene composition of the PaLoc showed 19 distinct genotypes among the 51 strains. Accordingly, 38 strains were classified mainly into 6 regular groups, while the remaining strains showed heterogeneous patterns. tcdC-/tcdD- constituted the most common genotypic group among the strains with partial PaLoc (7/51, 13.7%). A hypertoxigenic genotype, tcdC-/tcdA+/tcdB+, was detected in 2 strains (2/51, 3.9%). The intact genotype was also detected in a C. difficile isolate from outpatients. Cdt encoding genes toxins was observed in low numbers of the strains (7/52, 13.5%). All of cdtA+B+ strains were belonged to PaLoc group 1 (intact genotype). Statistical analyses showed no correlation between particular genotypes and special wards of the hospitals (p value>0.05). CONCLUSION: Collectively, our results showed diversity of C. difficile strains in most wards of the studied hospitals. Diversity of PaLoc genotypes in the strains that isolated from the same wards proposed endogenous routes of the infection, as common cause of CDI in these patients.

Truncated Core/NS3 Fusion Protein of HCV Adjuvanted with Outer Membrane Vesicles of Neisseria meningitidis Serogroup B: Potent Inducer of the Murine Immune System

Background: A licensed vaccine against hepatitis C virus (HCV) has not become available to date. The stability and antigenicity of a targeted synthesized recombinant fusion protein consisting of a truncated core and NS3 (rC/N) of HCV had been predicted. Although safe antigens, recombinant proteins are not efficacious vaccines without adjuvants. The present study evaluated the immunogenicity of rC/N as a bipartite antigen accompanied by Neisseria meningitidis serogroup B outer membrane vesicles (NMB OMVs) in BALB/c mice. Methods: The NMB OMVs were produced and evaluated accurately. The administrations were as follows: rC/N-OMV, rC/N-Freund's complete/incomplete adjuvant (CIA), rC/N-MF59, rC/N, OMV, MF59, and PBS. The production of Th1 (IFN-γ, IL-2)/Th2 (IL-4)/Th17 (IL-17) cytokines and granzyme B (cytotoxic indicator) by splenic mononuclear cells and the humoral concentration of total IgG/IgG1 (Th2)/IgG2a (Th1) in sera of mice were measured using mouse ELISA kits. Results: Concentrations of Th1/Th2/Th17 cytokines, granzyme B, and immunoglobulins in the spleens and sera of immunized mice, which had received antigen plus each adjuvant (rC/N-OMV, rC/N-Freund's CIA, and rC/N-MF59), significantly raised compared to the controls (rC/N, OMV, MF59, and PBS). Th1-type responses were dominant over Th2-type responses in vaccinated mice with rC/N-OMV, and Th2 type responses increased dominantly in vaccinated mice with rC/N-MF59 (p < 0.05). Discssion: NMB OMVs were able to increase Th1 immune responses dramatically more than MF59 and Freund's CIA. The formulation of rC/N with NMB OMVs showed its ability to induce Th1, Th2, and Th17 immune responses. rC/N-NMB OMVs is a promising approach for the development of an HCV therapeutic vaccine.

[Serotype distribution and antimicrobial resistance rates of Shigella spp. isolates in Tehran, Iran]. / Tahran'da izole edilen Shigella türlerinin serotip dagilimi ve antimikrobiyal direnç oranlari.

In this study, antimicrobial resistance patterns and serotype distributions of Shigella spp. isolated from pediatric and adult patients with diarrhea, inhabiting in Tehran, were investigated. Stool specimens of 1350 patients with diarrhea who were admitted to the seven different hospitals in Tehran from November 2003 till March 2005 were taken into the study. Antibacterial susceptibility patterns of Shigella spp. isolates were determined by standard disk diffusion method. Overall isolation rate of Shigella spp. was found as 11.5 percent. S. sonnei was the most frequent species (55.1%) followed by S. flexneri (30.8%), S. boydii (9.6%) and S. dysenteria (4.5%). Resistance rates to ampicillin (81.4%), trimethoprim-sulphamethoxazole (93.6%), chloramphenicol (28.2%) and tetracycline (98.7%) were high, whereas low resistance rates to cefixime (5.1%) and nalidixic acid (2.6%) were detected. All isolates were found susceptible to ceftriaxone and ciprofloxacine. These data may help physicians for choosing appropriate empirical chemotherapy although subsequent antibacterial susceptibility testing is always recommended.

Antibacterial resistance patterns of extended spectrum ß-lactamase -producing enteropathogenic Escherichia coli strains isolated from children.

BACKGROUND AND STUDY AIM: This study aimed to determine the antibacterial resistance patterns of extended spectrum ß-lactamase (ESBL)-producing enteropathogenic Escherichia coli (EPEC) isolated from Iranian children and to investigate its genetic patterns. PATIENTS AND METHODS: 192 non-repeats EPEC isolates were collected from stool samples of the children with and without diarrhoea. The EPEC strains were isolated from 1355 stool specimens obtained from 247 children with diarrhoea (0-10 years old; mean age, 5.5 years) and 1108 children without any gastrointestinal symptoms (0-10 years old; mean age, 6.8 years) during the summer months in three Iranian provinces, Tehran, Ilam and Mazandaran. Strains biochemically identified as E. coli were selected and were identified by the presence of eaeA and bfpA as EPEC virulence genes. Antimicrobial susceptibilities were determined by disc diffusion method. The isolates were confirmed to be ESBL producers by the double disk synergy test (DDST). The ß-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA) and insertion sequence ISEcp1 were detected by PCR method. RESULTS: The highest antibiotic susceptibility was detected to imipenem (100%), followed by gentamicin (82.3%) and ciprofloxacin (79.2%). The highest resistance was detected to cefpodoxime (97.9%), trimethoprim (60.7%), and tetracycline (58.4%), respectively. Totally, 153 EPEC strains (79.7%) were ESBL-producing by DDST test. The PCR showed that 84 (43.8%) EPEC isolates were positive for ESBLs encoding genes. Among 153 ESBLs-producing EPEC, TEM was present in 9.2% of isolates. Also, CTX-M and SHV genes were detected in 7.2% and 7.8%, respectively. The SHV positive strains were associated with the highest resistance rate to tetracycline (56.5%), although the TEM and OXA were associated with the highest resistance rate to gentamicin (23.1%) and ciprofloxacin (21.4%). CONCLUSIONS: The study revealed that 79.7% of EPEC isolates from Iranian children were ESBL-producing and were comparable with the non ESBL-producing isolates regarding susceptibility to the antibiotics.

Molecular study of carbapenemase genes in clinical isolates of Enterobacteriaceae resistant to carbapenems and determining their clonal relationship using pulsed-field gel electrophoresis.

PURPOSE: Enterobacteriaceae is a large family of Gram-negative bacteria that are considered as normal gut flora. They are the most common human pathogens. The main objective of this study was to investigate the carbapenemase genes in clinical isolates of Enterobacteriaceae resistant to carbapenem antibiotics and determine their clonal relationship using pulsed-field gel electrophoresis (PFGE). METHODOLOGY: In the present study, bacteria were isolated and identified via conventional biochemical tests and API 20NE. Antibiotic susceptibility was evaluated by using the disc diffusion method and MIC was carried out using the E-test. Phenotypic determination of carbapenemases was performed by employing a modified Hodge test (MHT). Carbapenemase genes including IMP, VIM, KPC, NDM and OXA-48 were amplified by PCR. The relationships between their clonal types with l restriction enzyme were examined using PFGE. RESULTS: Out of 40 isolates that were resistant or moderately susceptible to carbapenem antibiotics, 29 (72.5 %) strains were positive for carbapenem enzymes phenotypically. Moreover, six isolates contained carbapenemase genes including IMP, VIM, NDM and OXA-48, but the KPC gene was not found in any of the isolates. PFGE results showed that E. coli strains in our area were clustered into eight pulsotypes (A-H), Klebsiella spp. isolates five pulsotypes (A-E) and Proteus spp. had two pulsotypes (A, B). The high resistance to antimicrobial agents in the A, B and F pulsotypes was attributed to E. coli clinical isolates. CONCLUSIONS: Our results could reflect some hospital multidrug-resistant strains in nosocomial infections. The widespread emergence of carbapenem-resistant isolates has caused increasing concern in recent years. Therefore, specific strategies should be designed and evaluated for the control of resistant strains.