The prevalence of aminoglycoside-modifying enzyme genes (aac (6′)-I, aac (6′)-II, ant (2″)-I, aph (3′)-VI) in Pseudomonas aeruginosa

INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (6')-II (36%) was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2")-I, aph (3')-VI, and aac (6')-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a signific ant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance

Detection of Intracellular Adhesion (ica) Gene and Biofilm Formation Staphylococcus aureus Isolates from Clinical Blood Cultures

Background: In fact the biofilms are composed of bacterial cells living inmulticellular structures such as tissues and organs embedded within a self-produced matrix of extracellular polymeric substance (EPS). Ability to attach and biofilm formation are the most important virulence factors Staphylococcus aureus isolates. The aims of this study were to detect intracellular adhesion (ica) locus and its relation to the biofilm formation phenotype in clinical isolates of S. aureus isolated from bloodcultures. Methods: A total of 31 clinical S. aureus isolates were collected from Loghman Hospital of Tehran, Iran. In vitro biofilm formation ability was determined by microliter tissue culture plates. All clinical isolates were examined for determination the ica locus by using PCR method. Results: Twelve (38.7%) of the isolates were strong biofilm producers. The results showed that 18(80.6%) of the isolates carried icaD gene, whereas the prevalence of icaA, icaB and icaC were 51.6%, 45.1% and 77.4% respectively. Conclusions: S. aureus clinical isolates have different ability to form biofilm. This may be caused by the differences in the expression of biofilm related genes, genetic make-up and physiological conditions

COMPARISON OF CULTURE WITH POLYMERASE CHAIN REACTION FOR DETECTION OFUREAPLASMA UREALYTICUM IN ENDOCERVICAL SPECIMENS

Background: Ureaplasma urealyticum   is known as a sexually transmitted agent, causing mainly urethritis, pelvic inflammatory disease, spontaneous abortion, pyelonephritis, infertility, stillbirth, low birth weight, neonatal meningititis, and neonatal pneumonia. U. urealyticum infections not only jeopardize fertility but also pose a risk for infertility treatment and resulting pregnancies. Diagnosis of U. urealyticum infections by bacterial conventional methods is very difficult. The aim of this study was to compare culture with polymerase chain reaction (PCR) to determine the prevalence of U. urealyticum in endocervical specimens from infertile women. Methods: 312 endocervical swab samples were taken from infertile women, and transported with mycoplasma transport media. The culture was done with liquid-solid methods. DNA was extracted by Cadieux method, and analyzed by PCR protocol with species-specific U4&U5 primers. Results: U. urealyticum was detected in 26.2% (82/312) of specimens by both culture and PCR methods. 12.5% (39/312) of samples were PCR positive as well as culture positive, 11.2% (35/312) were positive only by PCR, and 2.5%(8/312) were positive only by culture. Conclusion: A sensitivity of 90% and 57% was found for PCR and culture respectively. PCR is therefore sensitive and more rapid (<24 hour) than culture (2-5 days) for the detection of U. urealyticum in endocervical secretions

Comparison Of Culture With The Polymerase Chain Reaction For Detection Of Gennital Mycoplasma

Aim: Genital mycoplasmas are known as sexually transmitted agents, causing mainly urethritis, pelvic inflammatory disease, spontaneous abortion, pyelonephritis, infertility, still birth, low birth weight, neonatal meningititis, and neonatal pneumonia. Mycoplasma infections not only jeopardize fertility but also pose a risk for infertility treatment and resulting pregnancies. Diagnosis of genital mycoplasma infections by bacterial conventional methods is very difficult. The aim of this study was to comparison of culture with polymerase chain reaction (PCR) for to determine the prevalence of Ureaplasma urealyticum and Mycoplasma hominis in the endocervical specimens from infertile women. Methods: 312 endocervical swab samples were taken from infertile women, and transported with a mycoplasma transport media. The culture was done with liquid-solid method. DNA was extracted by Cadieux method, and analyzed by PCR protocol with species-specific primers. Results: Genital mycoplasmas were detected in 35.5%( 111/312) specimens by both culture and PCR methods.16%(50/312) samples were PCR positive as well as culture positive, 23%(72/312) were positive only by PCR, and 2.8%(9/312) were positive only by culture. The sensitivity of 91.8% and 53% were found for PCR and culture respectively. Of the 111 positive specimens, 59 (53%) were positive only for U. urealyticum, 29(26%) were positive only for M. hominis and 23(20.7%) presented both organisms. Conclusion: Because of the potential adverse effects of mycoplasmas on the success rate of highly specialized infertility treatment, and their causal roles in several maternal complications of pregnancy and in neonatal morbidity and mortality, the rapid detection of mycoplasmas by PCR in infertile women could be important and necessary. The increased sensitivity and shorter time requirement of PCR support its further development for the diagnosis of mycoplasmas infections

Selenium Nanoparticles Induce Potent Protective Immune Responses against Vibrio cholerae WC Vaccine in a Mouse Model

The aim of this study was to evaluate the efficacy of selenium nanoparticle (an immune booster) and naloxone (an opioid receptor antagonist) as a new adjuvant in increasing immune responses against killed whole-cell Vibrio cholerae in a mouse cholera model. The Se NPs were synthesized and characterized by UV-visible, DLS, and zeta potential analysis. The SEM image confirmed the uniformity of spherical morphology of nanoparticle shape with 34 nm in size. The concentration of the Se NPs was calculated as 0.654 μg/ml in the ICP method. The cytotoxic activity of Se NPs on Caco-2 cells was assessed by the MTT assay and revealed 82.05% viability of cells after 24 h exposure with 100 μg/ml of Se NPs. Female BALB/C mice were orally immunized three times on days 0, 14, and 28, and challenge experiments were performed on immunized neonates with toxigenic V. cholerae. Administration of Se NP diet led to significant increase in V. cholerae-specific IgG and IgA responses in serum and saliva and caused protective immunity and 83.3% survival in challenge experiment against 1 LD50 V. cholerae in a group receiving diet of Se NPs compared with other groups including Dukoral vaccine. The IL-4 and IL-5 were significantly increased in response to WC+daily diet of Se NPs with or without naloxone. Naloxone proved no effect on IL-4 and IL-5 increase and is proposed as null in the cytokine and antibody production process. These results reveal that daily diet of Se NPs could efficiently induce immune cell effectors in both humoral and mucosal levels

Molecular Diagnosis of Salmonella enterica and Shigella spp. in Stool Sampleof Children With Diarrhea in Tehran

Background: Diarrheal illnesses caused by Salmonella and Shigella spp remain a serious public health problem in industrializing countries, and are still an important cause of morbidity and mortality in developed as well as undeveloped countries. Rapid detection of agents that cause diarrhea may help prevent occurrence of outbreaks. Objectives: The aim of this study was to compare conventional biochemical tests, namely API 20E strip system and Polymerase Chain Reaction (PCR) for identification of Salmonella enterica and Shigella spp. isolated from stool samples of patients with diarrhea. Materials and Methods: Stool samples were collected from patients with diarrhea during a three-year period (2009-2012). Conventional biochemical tests were used for identification of Shigella spp. and Salmonella enterica isolates. API 20E strips and PCR methods with oligonucleotide primers specific for ipaH of Shigella genus and hilA of Salmonella enterica were used to confirm the identity of the isolates. Results: Of the 81 suspected S. enterica and 112 Shigella spp. identified by conventional biochemical tests, 77 and 105 were identified as S.enterica and Shigella spp. by API 20E, respectively. All of the isolates were assigned to bacterial species with 99.9% probability value. All of the 81 suspected Salmonella enterica isolates produced 784 bp amplification bands of hilA gene. Among the 112 Shigella isolates confirmed by PCR, 90 (80.35%) were positive for wbgZ and 14 (12.5%) were positive for rfc genes indicative of S. sonnei and S. flexneri , respectively. Conclusions: In conclusion, the results of this study suggest that the PCR amplification of hilA and ipaH is a promising method for identification of Salmonella enterica and Shigella spp. The outcomes of this study can help towards more accurate and easy screening oflarge population of patients with Salmonella enterica and Shigella spp infections

Biological Activity of Recombinant Accessory Cholerae Enterotoxin (Ace) on Rabbit Ileal Loops and Antibacterial Assay

Objective: Vibrio cholerae (V. cholerae) causes a potentially lethal disease named cholera. The cholera enterotoxin (CT) is a major virulence factor of V. cholerae. In addition to CT, V. cholerae produces other putative toxins, such as the zonula occludens toxin (Zot) and accessory cholera enterotoxin (Ace). The ace gene is the third gene of the V. cholerae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli (E. coli) and determination of some characteristics of the recombinant Ace protein.Materials and Methods: In this experimental study, the ace gene was amplified from V. cholerae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli (DH5 α) host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-β-D-galctoside (IPTG) at a different concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa).Results: The recombinant Ace protein with a molecular weight of 18 kDa (dimeric form) was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. cholerae polyclonal antibody. The Ace protein had antibacterial activity at a concentration of ≥200 μg/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test.Conclusion: This study described an E. coli cloning and expression system (E. coli BL21- pET-28a-ace) for the Ace protein of V. cholerae. We confirmed the antibacterial properties and enterotoxin activity of the resultant recombinant Ace protein

Cumulative protective efficacy of rZot and rAce combination in challenge experiments with wild type Vibrio cholerae in mouse model

The aim of this study was to assess the cumulative immunogenicity properties of rZot and rAce combination and their potential ability to increase the clearance rate of pathogenic standard Vibrio cholerae strain in challenge experiments in mice model. The recombinant Zot and Ace proteins were produced and used to raise polyclonal antibodies of anti-Zot and anti-Ace recombinant proteins in rabbit. Six-week female BALB/c mice were immunized with different antigens via oral route. Blood samples were collected, and the total amount of IgG and IgA antibodies against rZot and rAce were measured in blood and stool samples of each immunized mouse. Challenge experiments were done with toxigenic V. cholerae strain. The anti-Zot and anti-Ace IgG titers were significantly higher in immunized mice in comparison with control group. The IgG and IgA titers were higher in the sera of mice immunized by recombinant Ace than in group immunized by rZot, indicating the higher immunogenicity of rAce than rZot. The use of rAce and rZot mixture led to synergistic activities in increasing the level of IgG and IgA in comparison with the use of each protein separately. The clearance rate was significantly higher in different challenge groups than in the control group, and the coherence between rZot and rAce reduced the bacterial shedding significantly. In conclusion, the use of recombinant Zot and Ace mixture can produce the proper amount of IgA and IgG against to toxigenic V. cholerae, reduce bacterial shedding in immunized mice significantly, and be used as a potent candidate in cholera vaccine research

Cloning and Expression of Helicobacter pylori HpaA Gene

Objective: Helicobacter pylori is associated with chronic gastritis, peptic ulcers, gastric adenocarcinomaand gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Antibiotictherapies do not protect from potential re-infection and have a risk for development of drugresistance. Therefore, prophylactic vaccine mediated protection against H. pylori is an attractiveclinical interest. H. pylori adhesin A (HpaA) is a conserved surface lipoprotein and playsimportant roles in the pathogenesis of infection. In this study the recombinant protein (rHpaA)was over-expressed in E.coli.Materials and Methods: The hpaA gene was amplified by PCR. Prokaryote expression vectorpET28a-hpaA was constructed, and used to transform E.coli BL21DE3. The expressionof recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot wereused to determine immunoreactivity of rHpaA by a rabbit polyclonal antibodies against wholecell of H. pylori.Results: The hpaA gene nucleotide sequence in the recombinant plasmid vector of pET-28-a-hpaA was consistent with that of H.pylori hpaA as published in the GenBank. SDS-PAGEdemonstrated that the constructed prokaryotic expression efficiently produced rHpaA at the1.5 mmol/L of IPTG. HpaA fusion protein was able to react with the rabbit polyclonal antibodyagainst whole cells of H. pylori.Conclusion: A prokaryotic expression system pET-28a-hpaA-BL21 with high efficiency of H.pylori hpaA gene was successfully established and the HpaA fusion protein showed satisfactoryimmunoreactivity. These results indicate that production of a specific recombinant proteinis an alternative and potentially more expeditious strategy for development of H. pylori vaccine

Prevalence of Multidrug Resistance in Enterococcus faecium Isolated from Patients and Environment of Hospitals in Lorestan Province, (Iran)

Background and Objectives: With increasing use of vancomycin antibiotic, vancomycin-resistant Enterococcus (VRE), is considered as a major cause of nosocomial infections. In this study, the pattern of antibiotic resistance and prevalence of vancomycin resistant enterococcus faecium, were investigated. &nbsp; Methods: In this descriptive study, after isolation and identification of 690 strains of Enterococcus from clinical and environmental specimens in different wards of Lorestan hospitals, the pattern of antibiotic resistance of these strains to common antibiotics, were investigated using dick diffusion and agar dilution methods. The antibiotic resistance genes (vanA, vanB, pbp5, pbpZ, and blaZ), were detected by PCR technique. &nbsp; Results: Among 690 studied Enterococcus isolates, 439 isolates (64%), were Enterococcous feacalis, 228 isolates (33%) Enterococcous faecium, and 23 isolates (3%), were other Enterococci. Based on the antibiogram test by disk diffusion method, the highest resistance was to penicillin with 446 isolates (67%) and the lowest resistance was to linezolid with 0 isolate (0%). In the evaluation of resistance using minimum inhibitory concentration (MIC), the antibiotic resistance rate was obtained to be 58% for ampicillin, 59% for penicillin, and 23% for vancomycin. Resistance genes, including vanA, vanB, pbp5, pbpZ, and blaZ, were detected in 84 (72%), 26 (22%), 87 (74%), 63 (53%), and 0 (0%) of the isolates. &nbsp; Conclusion: Based on the results of this study, resistance to vancomycin in Enterococcus strains is also found in Lorestan province like other parts of the world. Therefore, application of control and preventive measures are necessary. Furthermore, to prescribe appropriate antibiotic, it is recommended to perform antibiogram test for each patient before the treatment. &nbsp; &nbsp