Investigating of four main carbapenem-resistance mechanisms in high-level carbapenem resistant Pseudomonas aeruginosa isolated from burn patients.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen involved in many infections. Carbapenem-resistant P.aeruginosa has emerged as an important cause of infection in different hospitals worldwide. We aimed to determine frequencies of the four main resistance mechanisms [metallo-beta lactamase (MBL) production (blaIMP, blaVIM, blaSPM and blaNDM), overproduction of the MexAB-OprM and MexXY efflux pumps, overproduction of chromosome-encoded AmpC ß-lactamase, and reduced OprD expression] in high-level carbapenem-resistant P.aeruginosa isolated from patients with burns. METHODS: In a descriptive study, 107 P. aeruginosa isolates were collected from patients with burn injuries and tested for antibiotic susceptibility, by an E-test for carbapenems, an E-test for metallo-ß-lactamase producer isolates, and PCR to detect MBL genes. Furthermore, high-level carbapenem-resistant isolates were tested by real-time PCR for the expression levels of the mexB, mexY, ampC, and oprD genes. RESULTS: Amongst all P. aeruginosa isolates, 78.5%, 46.7%, and 15% were imipenem-, meropenem-, and doripenem-resistant, respectively; 72% of isolates were multidrug-resistant. The blaIMP and blaVIM genes were detected in 17.9% and 1.2% of isolates; respectively. The blaSPM and blaNDM genes were not observed. Among the resistant isolates, mexB overexpression (63.2%) was the most frequent mechanism, followed by mexY overexpression (52.6%), ampC overexpression (36.8%), and reduced oprD expression (21.1%). CONCLUSION: Emerging antimicrobial resistance in burn wound bacterial pathogens is a serious therapeutic challenge for clinicians. In the present study, most of the isolates were MDR. This finding indicated an alarming spread of resistant isolates and suggested that infection control strategies should be considered. Resistance to carbapenems is influenced by several factors, not all of which were evaluated in our study; however, the results showed that production of MBLs and overexpression of the mexB gene were the most frequent mechanisms in carbapenem-resistant isolates.

Detection of Theileria lestoquardi cross infection in cattle with clinical theileriosis in Iran.

Theileriosis caused by Theileria lestoquardi (malignant ovine theileriosis) in sheep and Theileria annulata (tropical theileriosis) in cattle is an important hemoprotozoal tick-borne disease in Iran. Due to major biologic and phylogenic similarities of these two species, this study was carried out to investigate the occurrence of natural infections with T.lestoquardi and T.annulata in cattle with clinical theileriosis in Ahvaz, southwest Iran. Fifty one cattle were selected based on clinical signs of theileriosis and confirmation by microscopic examination of blood smears. Blood samples were collected from each animal and hematologic and microscopic examinations were performed. Theileria piroplasmic forms were detected in all affected cattle. Pale mucous membranes (43.14%), icterus (11.76%) and fever (70.6%) were also observed. PCR-RFLP analysis revealed T. annulata infection in all tested cattle while coinfections with T. lestoquardi were found in two samples (3.92%). All sampled cattle including the two with mixed species Theileria infection were anemic. This is the first report of Theileria species cross infections in cattle with clinical theileriosis in Iran. It can be concluded that cattle can be infected with both pathogenic Theileria species, T. lestoquardi and T. annulata which can be an important issue in the epidemiology and spread of ovine malignant theileriosis.

Molecular cloning of an alpha-enolase from the human filarial parasite Onchocerca volvulus that binds human plasminogen.

Enolase represents a multifunctional protein involved in basic energy metabolism and plasminogen binding and activation at the surface of prokaryotic pathogens. A complete cDNA of 1615 bp of an alpha-enolase from Onchocerca volvulus (Ov-ENO) was isolated using a PCR-based approach. The open reading frame encoded for 435 amino acids and the high degree of conservation included the crucial amino acid residues that participate in the formation of the catalytic site, Mg(2+) binding site, and a hydrophobic motif reported to relate to surface expression. A 1089-bp fragment was expressed in a N-terminal 6 x His-tag expression vector in Escherichia coli. By immunohistological analysis using anti-Ov-ENO rabbit antibodies, native enolase could be detected in most tissues of adult O. volvulus, microfilariae, and infective larvae. Intense staining was observed in the muscles, where the energy consumption is high. The purified recombinant protein fragment revealed plasminogen binding activity in a blot-overlay assay employing anti-plasminogen antibodies. In sera from individuals infected with O. volvulus, IgG antibodies reactive with recombinant Ov-ENO were demonstrated by immunoblot and enzyme-linked immunosorbent analyses. The plasminogen-binding property of O. volvulus alpha-enolase may support plasmin-mediated proteolysis including degradation of host's extracellular matrix thereby promoting the migration of larval stages through tissues. The recognition by antibodies in sera of O. volvulus-infected persons indicate an involvement of the protein in the interaction between the parasite and the human host.

Identification of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from burn patients by multiplex PCR.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) as important human pathogens are causes of nosocomial infections worldwide. Burn patients are at a higher risk of local and systemic infections with these microorganisms. OBJECTIVE: A screening method for MRSA by using a multiplex polymerase chain reaction (PCR) targeting the 16S ribosomal RNA (rRNA), mecA, and nuc genes was developed. The aim of the present study was to investigate the potential of this PCR assay for the detection of MRSA strains in samples from burn patients. METHODS: During an 11-month period, 230 isolates (53.11%) of Staphylococcus spp. were collected from burn patients. The isolates were identified as S. aureus by using standard culture and biochemical tests. DNA was extracted from bacterial colonies and multiplex PCR was used to detect MRSA and MRCoNS strains. RESULTS: Of the staphylococci isolates, 149 (64.9%) were identified as S. aureus and 81 (35.21%) were described as CoNS. Among the latter, 51 (62.97%) were reported to be MRCoNS. From the total S. aureus isolates, 132 (88.6%) were detected as MRSA and 17 (11.4%) were methicillin-susceptible S. aureus (MSSA). The presence of the mecA gene in all isolates was confirmed by using multiplex PCR as a gold standard method. CONCLUSION: This study presented a high MRSA rate in the region under investigation. The 16S rRNA-mecA-nuc multiplex PCR is a good tool for the rapid characterization of MRSA strains. This paper emphasizes the need for preventive measures and choosing effective antimicrobials against MRSA and MRCoNS infections in the burn units.

Wolbachia endosymbionts of Onchocerca volvulus express a putative periplasmic HtrA-type serine protease.

Wolbachia are intracellular bacteria of many filarial nematodes. A mutualistic interaction between the endobacteria and the filarial host is likely, because the clearance of Wolbachia by tetracycline leads to the obstruction of embryogenesis and larval development. Databases were searched for exported molecules to identify candidates involved in this mutualism. Fragments of a Wolbachia serine protease from the human filarial parasite Onchocerca volvulus were obtained (Wol-Ov-HtrA) by the use of a PCR technique and primers based on the Rickettsia prowazekii genome. The deduced amino acid sequence exhibited 87% and 81% identity to the homologous Wolbachia proteases identified from Brugia malayi and Drosophila melanogaster, respectively. The full-length cDNA encodes 494 amino acids with a calculated mass of 54 kDa. Three characteristic features, (i) a catalytic triad of serine proteases, (ii) two PDZ domains and (iii) a putative signal peptide, classify the endobacterial protein as a member of the periplasmic HtrA family of proteases known to express chaperone and regulator activity of apoptosis. Using a rabbit antiserum raised against a recombinantly expressed 33-kDa fragment of Wol-Ov-HtrA, strong labelling of the antigen was found associated with endobacteria in hypodermis, oocytes, zygotes, all embryonic stages and microfilariae of O. volvulus. Staining of hypodermal cytoplasm surrounding the endobacteria indicated a possible release of the protein from the Wolbachia. The demonstration of Wol-Ov-HtrA-reactive IgG1 antibodies in sera of O. volvulus-infected persons indicated the exposure to the protein and its recognition by the human immune system. Wol-Ov-HtrA is a candidate for an exported Wolbachia protein that may interact with the filarial host metabolism.

Isolation and identification of excretory-secretory and somatic antigens from the Oestrus ovis larvae by SDS-PAGE and immunoblotting.

Oestrus ovis is an economically important parasite of small ruminants and a zoonotic parasite with many reports of ophthalmomyiasis in human from Iran and other countries. The aim of the peresent study was the isolation and identification of excretory-secretory (ES) and somatic (S) antigens of O. ovis second and third stage larvae (L2, L3) collected from Arabi sheep breeds located in southwest of Iran. Positive sera were prepared by marking the sheep, taking blood sample and direct observation of the parasite in the head. Somatic antigens of the larvae (SL2, SL3) were prepared by sonication. Larval excretory-secretory antigens (ESL2, ESL3) were prepared by incubation the larvae in RPMI-1640 RPMI medium. Electrophoretic protein profiles of ESL2 two, ESL3 seven, SL2 eight, SL3 fifteen bands (from 79.0 to below 14.4 KDa) were shown. In immunoblotting with positive sera, four common bands in SL2 and SL3 at 58, 42.0, 29.0 and 28.0 kDa, one specific band in SL3 at 47.0 kDa and one band in ESL2, at 28.0 kDa, and three bands in ESL3 at 58.0, 42.0, 29.0 and 28.0 kDa were recognized. Among the profiles, the 28 kDa protein was the most common antigenic component. Nevertheless, the antigenic proteins 29, 58 kDa were a common protein in electrophoretic patterns of both S and ES proteins of L2 and L3 but, 42.0 kDa antigen the only one detected in immunoblot but not in S and ES protein profiles of the larvae. Therefore, the antigens 29.0, 42.0 and 58.0 kDa can be used for further studies of protective effects and serological diagnostic methods.

Vertical transmission of Theileria lestoquardi in sheep.

This is the first report of an outbreak of Theileria lestoquardi abortion and stillbirth in a mob of 450 ewes in July 2012, during which, approximately 35 late-term ewes lost their fetuses over a 5-day period. A dead ewe and her aborted fetus were transported to the Ahvaz Veterinary Hospital for the diagnostic evaluation. The microbial cultures from the ewe vaginal discharges and fetal abomasal contents and the liver were negative. The blood films of the ewe and her fetus contained Theileria piroplasms and the impression smears from ewe liver and fetal spleen were positive for Theileria Koch blue bodies. The DNA was extracted from the liver and spleen of ewe and her fetus, respectively, and analyzed by polymerase chain reaction (PCR) using specific primers derived from the nucleotide sequences of 18S ribosomal DNA (rDNA) gene of T. lestoquardi. A single fragment of 428-bp fragment was amplified. The PCR product was directly sequenced and the alignment of the sequence with similar sequences in GenBank(®) showed 100% identities with 18S rDNA gene of T. lestoquardi. The present study is the first report of the T. lestoquardi vertical transmission that could be related to the abortion.

Production of Recombinant Alpha Neurotoxin of Scorpion Venom Mesobuthus eupeus and Analysis of its Immunogenicity.

BACKGROUND: Scorpion venom is important and rich source of peptides, most of which have been widely used as pharmacological tools for unraveling structure-function relationship of various ion channels. Naturally occurring toxins can be also considered as lead compounds in the development of novel drugs. OBJECTIVES: In this context, the scorpion-derived peptide neurotoxins specific to sodium channels have shown promise as potential therapeutic targets for the treatment of various human diseases. MATERIALS AND METHODS: A cDNA library from the extracted RNA was constructed using RT-PCR and semi-nested RT-PCR. DNA sequencing followed by phylogenetic analysis was applied to screen the cDNA library clones. For molecular characterization of the BMK gene we used cloning and recombinant protein expression techniques based on E.coli systems. Then we performed mice immunization and Western blot and Immunodot analyses. RESULTS: A novel BMK neurotoxin has been cloned, expressed and characterized from the Iranian scorpion M. eupeus venom. We analyzed the recombinant BMK by immunoblotting with treated antiserum. The result showed that mice antiserum can react also with scorpion crude venom, so is able to recognize native BMK toxin. CONCLUSION: The newly produced recombinant protein BMK revealed to be immunogenic. Moreover, anti-BMK antibodies produced in mice were able to recognize both the recombinant BMK neurotoxin and the one in M. eupeus crude venome. Taken together, the molecular characterization and recombinant production of the Iranian scorpion M. eupeus venom component can serve as a new probe for further studies of sodium channels function and physiology. This provides a promising perspective for the future design of selective drugs, as well as for research of antivenom production.

Application of Pulsed Field Gel Electrophoresis for Study of Genetic Diversity in Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients.

BACKGROUND: Mycobacterium tuberculosis genotyping can effectively improve tuberculosis (TB) control programs by controlling disease transmission. Pulsed field gel electrophoresis (PFGE) is a particularly powerful tool for determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease. OBJECTIVES: The aim of present study was to investigate the genetic diversity of M. tuberculosis strains in Khuzestan province by the PFGE technique. PATIENTS AND METHODS: In total, 80 M. tuberculosis positive cultures were obtained from tuberculosis patients. PFGE was performed on 60 PCR-confirmed isolates by using DraI and XbaI restriction enzymes according to standard protocols. Plugs containing digested DNA were then loaded on agarose gels and run using contour-clamped homogenous electric fields. RESULTS: Fifty distinct DNA banding patterns were obtained by digestion of DNA with DraI and 38 DNA banding patterns by digestion with XbaI restriction enzymes. The patterns comprised of 17 different clusters in which cluster I was the major one, containing six strains. Three clusters contained three strains each and the 13 remaining clusters comprised of two strains each. Digestion with DraI yielded 15-20 DNA fragments with 50-485 kb size, while digestion by XbaI produced DNA fragments with a size smaller than 50-242 kb. CONCLUSIONS: Despite the ability of PFGE for study of genetic diversity of many mycobacterial species and it being considered as a robust and useful tool, in this study we only found a 15% epidemiological relationship amongst the isolates. Thus, for higher discrimination of genotypic clusters among M. tuberculosis clinical isolates, the application of more sophisticated complementary techniques is required.

Detection of Metallo-Beta Lactamases Among Carbapenem-Resistant Pseudomonas aeruginosa.

BACKGROUND: Carbapenems are important drugs used for the treatment of Pseudomonas aeruginosa infections, however metallo-ß-lactamases (MBL) are able to efficiently hydrolyze these classes of drugs. Immediate detection of the MBL-producing P. aeruginosa is necessary in order to accurately treat infections caused by this organism. OBJECTIVES: To determine the prevalence of MBL producing P. aeruginosa in burn and non-burn patients by two phenotypic tests and polymerase chain reaction (PCR) and to compare phenotypic tests with PCR. MATERIALS AND METHODS: A total of 223 non-duplicate strains of P. aeruginosa were collected from three teaching hospitals of Ahvaz, Iran. Antimicrobial susceptibility and minimum inhibitory concentrations (MICs) of carbapenems (imipenem, meropenem, doripenem and ertapenem) were determined by the Kirby-Bauer and E-test methods. Combined disk (CD) test, MBL E-test and PCR were performed for carbapenem-resistant P. aeruginosa isolates. RESULTS: Amongst all the P. aeruginosa isolates, 58.7% were resistant to imipenem while 31.8%, 13.5% and 74.4% were resistant to meropenem, doripenem and ertapenem, respectively. Amongst all the P. aeruginosa isolates, 44.4% were multidrug resistant and 13.45% were resistant to all of the carbapenems. The CD test with doripenem disk / 750 µg ethylene diamine tetra acetic acid (EDTA) had the highest efficiency compared to the other phenotypic tests. bla IMP and bla VIM genes were detected in 11.7% and 0.4% of isolates, respectively. bla SPM and bla NDM genes were not observed. CONCLUSIONS: Epidemiological and regional evaluation of MBL-producing P. aeruginosa through simple and inexpensive methods should be considered for effective treatment of carbapenem-resistant P. aeruginosa infections.

Sequence characterization of cDNA sequence of encoding of an antimicrobial Peptide with no disulfide bridge from the Iranian mesobuthus eupeus venomous glands.

BACKGROUND: Scorpion venom glands produce some antimicrobial peptides (AMP) that can rapidly kill a broad range of microbes and have additional activities that impact on the quality and effectiveness of innate responses and inflammation. OBJECTIVES: In this study, we reported the identification of a cDNA sequence encoding cysteine-free antimicrobial peptides isolated from venomous glands of this species. MATERIALS AND METHODS: Total RNA was extracted from the Iranian mesobuthus eupeus venom glands, and cDNA was synthesized by using the modified oligo (dT). The cDNA was used as the template for applying Semi-nested RT- PCR technique. PCR Products were used for direct nucleotide sequencing and the results were compared with Gen Bank database. RESULTS: A 213 BP cDNA fragment encoding the entire coding region of an antimicrobial toxin from the Iranian scorpion M. Eupeus venom glands were isolated. The full-length sequence of the coding region was 210 BP contained an open reading frame of 70 amino with a predicted molecular mass of 7970.48 Da and theoretical Pi of 9.10. The open reading frame consists of 210 BP encoding a precursor of 70 amino acid residues, including a signal peptide of 23 residues a propertied of 7 residues, and a mature peptide of 34 residues with no disulfide bridge. The peptide has detectable sequence identity to the Lesser Asian mesobuthus eupeus MeVAMP-2 (98%), MeVAMP-9 (60%) and several previously described AMPs from other scorpion venoms including mesobuthus martensii (94%) and buthus occitanus Israelis (82%). CONCLUSIONS: The secondary structure of the peptide mainly consisted of α-helical structure which was generally conserved by previously reported scorpion counterparts. The phylogenetic analysis showed that the Iranian MeAMP-like toxin was similar but not identical with that of venom antimicrobial peptides from lesser Asian scorpion mesobuthus eupeus.

Effects of Teucrium polium Ethyl acetate Extract on Serum, Liver and Muscle Triglyceride Content of Sucrose-Induced Insulin Resistance in Rat.

Possessing putative hypolipidemic effects, Teucrium polium (TP) have been traditionally used as a medicinal plant in Iran. The aim of the present study was to investigate this effect on the sucrose-induced insulin resistance male rat model. Thirty Wistar male rats weighting 180 ± 20 g were divided into five groups of six each. Four groups were given sucrose 50% in drinking water for 10 weeks. In 8(th) week of treatment, three groups of them were randomly selected and treated with Teucrium polium (T. polium) ethyl acetate extract (50, 100 and 200 mg/Kg for two weeks). Control animals were fed using normal rat chow. After ten weeks, blood samples were collected from the heart. Blood Glucose, insulin, leptin, lipid content and fasting insulin resistance index (FIRI) as well as liver and muscle glycogen and lipid contents were determined. Final data were analyzed by ANOVA and post-hoc Tukey's test. Liver glycogen contents and blood levels of glucose and insulin were significantly increased in high sucrose (HS) group compared with control group. A significant decrease was observed in blood glucose and insulin levels, FIRI, serum total lipid, triglyceride and VLDL-c as well as the liver triglyceride level, muscle and liver glycogen contents in 100 and 200 mg/Kg of TP-treated groups compared with HS group. Leptin level was significantly decreased in 50 and 100 mg/Kg groups compared with HS group. The treatment with T. polium ethyl acetate extract (TP-EAE) induced a dose-dependent reduction in serum, liver and muscle triglyceride (TG) and liver glycogen content levels, as well as serum insulin. These effects may be attributed, in part, to the hypolipidemic effect of TP flavonoids; otherwise, the hepatoprotective and antioxidant activity of TP-EAE may improve the liver function and reverse harmful sucrose effects.

Onchocerca volvulus: expression and immunolocalization of a nematode cathepsin D-like lysosomal aspartic protease.

The N-terminal region of the cathepsin D-like aspartic protease from the human filarial parasite Onchocerca volvulus was expressed as His-tag fusion protein. Light and electron microscopic immunohistology using antibodies against the recombinant protein showed labeling of lysosomes in the hypodermis and epithelia of the intestine and the reproductive organs of Onchocerca. While developing oocytes were negative, mature oocytes and early morulae showed strong labeling. In older embryos and mature microfilariae, stained lysosomes were only found in a few cells. Cell death in degenerating microfilariae of patients untreated and treated with microfilaricidal drugs was associated with strong expression of aspartic protease. IgG1, IgG4, and IgE antibodies reactive with the recombinant protein were demonstrated in sera from onchocerciasis patients indicating exposure and recognition of the enzyme by the host's defence system. The aspartic protease of O. volvulus appears to function in intestinal digestion and tissue degradation of the filaria.

Construction and expression of aspartic protease from Onchocerca volvulus* as ompA fusion protein in a mutant strain of Salmonella typhimurium

Two constructions in pHS164 vector were designed to permit expression of OV7A and OV4A inserts encoding the N-terminal and C-terminal portion of an aspartic protease from Onchocerca volvulus, respectively. A novel 39 kD protein ompA-OV7A fusion protein was stably expressed as ompA fusion in a modified strain of Salmonella typhimurium strain SL5000 and E.coli strain JM109. Expression of the fusion protein in bacterial strains harboring the constructs were evaluated by western blotting. E.coli and Salmonella lysates were fractionated by 10 percent SDS-PAGE gel and then immobilized to nitrocellulose membrane by electroblotting. Primary polyclonal antibody generated in rats against the GST-OV7A fusion protein was used in the Western blots. It remains to be seen whether the fusion protein expressed in vivo will promote effective immune response