Rapid and direct molecular detection of Streptococcus pneumoniae and Haemophilus influenzae isolated in oropharynx and nasal cavity of children.

There is a direct correlation between asymptomatic oropharyngeal colonization by pathogenic bacteria and the prevalence of paediatric respiratory infections. Streptococcus pneumoniae and Haemophilus influenzae are common cause of serious bacterial infections such as meningitis in children. This study aimed to monitor healthy children to detect asymptomatic carriers of S. pneumoniae and H. influenzae. In the present cross-sectional study, real-time PCR assay was developed and evaluated in comparison with culture for direct detection of S. pneumoniae and H. influenzae in 123 oropharynx and nasal cavity specimens from healthy children in Ilam, Iran. In addition, virulence factor (ply and hpd) and iron uptake (tbpA and piuA) genes were evaluated by PCR. Our results demonstrated that among all isolates only 14 S. pneumoniae and eight H. influenzae were identified by phenotypic methods, whereas 37 and 21 S. pneumoniae and H. influenzae were identified, respectively, by real-time PCR assay. All S. pneumoniae and H. influenzae isolates possessed tbpA and piuA genes. Also, 81% (30/37) of S. pneumoniae isolates were positive for ply gene, which encoded pneumolysin, as well as 90% (19/21) of H. influenzae isolates were positive for hpd gene. Simultaneous colonization of S. pneumoniae and H. influenzae could indicate the importance of monitoring of healthy children to identification of carriers.

Mutation and Efflux System are Responsible for Ciprofloxacin Resistance in Klebsiella pneumoniae / Las mutaciones y el sistema de eflujo son responsables de la resistencia a la ciprofloxacina en la Klebsiella pneumoniae

ABSTRACT The main mechanism of quinolone resistance in Klebsiella (K) pneumoniae is caused by mutation of porin-related proteins and efflux pumps. This study aimed to investigate the prevalence of ciprofloxacin-resistant K pneumoniae in burns patients and to understand the role of the AcrAB multidrug efflux system on minimal inhibitory concentration (MIC) of ciprofloxacin. For this reason, 52 K pneumoniae samples were collected from burns patients and evaluated for the mechanism of ciprofloxacin resistance. The results demonstrated that 40 isolates of K pneumoniae were ciprofloxacin-resistant and 35 showed the mutation on gyrA locus. By inhibition of the efflux system, the MIC yield showed a significant decrease. Therefore, it could be concluded that the high rate of mutation on the gyrA locus in combination with quinolone resistance was responsible for ciprofloxacin resistance and by inhibition of AcrA, the resistance rate showed a significant decrease in K pneumoniae isolated from burns patients.

RESUMEN El principal mecanismo de resistencia a la quinolona en las Klebsiella (K) Pneumoniae tiene como causa la mutación de las porinas y las bombas de eflujo. Este estudio tuvo por objetivo investigar la prevalencia de las K pneumoniae resistentes a la ciprofloxacina en pacientes con quemaduras, así como entender el papel del sistema de eflujo multidroga AcrAB en la concentración inhibitoria mínima (CIM) de la ciprofloxacina. Por esta razón, se recogieron 52 muestras de K pneumoniae de pacientes con quemaduras, a fin de evaluar el mecanismo de resistencia a la ciprofloxacina. Los resultados mostraron que 40 aislados de K pneumoniae eran resistentes a la ciprofloxacina y 35 mostraron la mutación en el locus gyrA. Con la inhibición del sistema de eflujo, el rendimiento de CIM tuvo una disminución significativa. Por lo tanto, se pudo concluir que la alta tasa de mutación en el locus gyrA en combinación con la resistencia a la quinolona era responsable de la resistencia a la ciprofloxacina, y por la inhibición de AcrA, la tasa de resistencia mostró una disminución significativa en las K pneumoniae aisladas de los pacientes con quemaduras.

Simultaneous lactic acidification and coagulation by using recombinant Lactococcus lactis strain.

AIMS: This study was an attempt to create a novel milk clotting procedure using a recombinant bacterium capable of milk coagulation. METHODS AND RESULTS: The Rhizomucor pusillus proteinase (RPP) gene was sub-cloned into a pALF expression vector. The recombinant pALF-RPP vector was then electro-transferred into Lactococcus lactis. Finally, the milk coagulation ability of recombinant L. lactis carrying a RPP gene was evaluated. Nucleotide sequencing of DNA insertion from the clone revealed that the RPP activity corresponded to an open reading frame consisting of 1218 bp coding for a 43·45 kDa RPP protein. The RPP protein assay results indicated that the highest RPP enzyme expression with 870 Soxhlet units (SU) per ml and 7914 SU/OD were obtained for cultures which were incubated at pH 5·5 and 30°C. Interestingly, milk coagulation was observed after 205 min of inoculating milk with recombinant L. lactis carrying the RPP gene. CONCLUSION: The recombinant L. lactis carrying RPP gene has the ability to function as a starter culture for acidifying and subsequently coagulating milk by producing RPP as a milk coagulant agent. SIGNIFICANCE AND IMPACT OF THE STUDY: Creating a recombinant starter culture bacterium that is able to coagulate milk. It is significant because the recombinant L. lactis has the ability to work as a starter culture and milk coagulation agent.

Ex Vivo Evaluation of Thymus daenensis as an Antioxidant and Antibacterial Medicinal Herb.

Herbal medicines are defined as traditionally used natural products. The current study in an attempt try to investigate the antibacterial activity on extended spectrum beta-lactamases (ESBL), methalo beta-lactamases (MBL) producing gram negative bacteria and methicillin resistant Staphylococcus aureus (MRSA) and subsequently, to determine the antioxidant activity of Thymus daenensis. For this reasons, firstly cytotoxicity of T. daenensis was determined by MTT assay. Then, essential oil was subjected for antibacterial and antioxidant activity. Our results demonstrated that 15 mg/ml concentration of T. daenensis inhibited both P. aeruginosa producing MBL and E.coli producing ESBL, while this value was 25 mg/ml concentration for MRSA inhibition. The association between phenolic compound and antioxidant activity was found for the ABTS•+ method (43.52%) in the lowest level, while, for FRAD and DPPH• methods the opposite story occurred (70.5% correlation for DPPH• and 50.9% for FRAD). Our findings suggested that T. daenensis has a potential herbal medicine that should be considered as an antibacterial and antioxidant with very low toxicity.

The rationale behind antibiotic resistance pattern in Klebsiella pneumoniae.

Presently, there is an increase in antibiotic resistance in bacteria, due to relax prescription of antibiotics, especially in Iran. Undoubtedly, in toxin antitoxin (TA) system, a toxin neutralized by antitoxin, which known as a potent antimicrobial target; but there is no extensive survey on the prevalence of TA loci in large scale of Klebsiella pneumoniae. Therefore, this study aims to determine the prevalence of different TA loci in clinical and environmental K. pneumoniae isolates. For this reason, 48 K. pneumoniae clinical isolates and 49 K. pneumoniae environmental isolates were subjected for evaluation of different TA loci. The results of current study indicated that there is no association between antibiotic resistances and presence of TA loci in clinical and environmental K. pneumoniae. The role of TA loci as a potent target in antibiotic resistant K. pneumoniae has been complicated. Therefore, more studies should be performed to explain why TA loci are presented in K. pneumoniae and what is the rationale behind antibiotic resistant K. pneumoniae?

The rationale behind antibiotic resistance pattern in Klebsiella pneumoniae

Presently, there is an increase in antibiotic resistance in bacteria, due to relax prescription of antibiotics, especially in Iran. Undoubtedly, in toxin antitoxin (TA) system, a toxin neutralized by antitoxin, which known as a potent antimicrobial target; but there is no extensive survey on the prevalence of TA loci in large scale of Klebsiella pneumoniae. Therefore, this study aims to determine the prevalence of different TA loci in clinical and environmental K. pneumoniae isolates. For this reason, 48 K. pneumoniae clinical isolates and 49 K. pneumoniae environmental isolates were subjected for evaluation of different TA loci. The results of current study indicated that there is no association between antibiotic resistances and presence of TA loci in clinical and environmental K. pneumoniae. The role of TA loci as a potent target in antibiotic resistant K. pneumoniae has been complicated. Therefore, more studies should be performed to explain why TA loci are presented in K. pneumoniae and what is the rationale behind antibiotic resistant K. pneumoniae?

BERBERIS VULGARIS FRUIT CRUDE EXTRACT AS A NOVEL ANTI-LEUKAEMIC AGENT.

Tumor protein p53 encoded by the TP53 gene in humans is known as a cancer biomarker in patients diagnosed with cancer, and it plays an essential role in apoptosis, genomic stability, and inhibition of angiogenesis. Cancer therapies with common chemotherapy methods are effective, as known, but have some side effects. Berberis vulgaris is traditionally administrated as a cancer drug. The current research aims to evaluate p53 as a biomarker in WEHI-3 cell line and to demonstrate the Berberis vulgaris fruit crude extract (BVFCE) as a new anticancer drug. For this purpose, we evaluated the effect of BVFCE in different concentrations against WEHI-3cell line in vitro and determined the quantitative level of p53 gene in the treated WEHI-3 cells. The results demonstrated that even at only 1 mg/ml concentration of Berberis vulgaris crude extract, there was a low level of p53 biomarker expression on WEHI-3 cells in comparison with doxorubicin. Therefore, the current study suggests BVFCE as a reliable anti-leukaemic drug and candidate for anticancer therapy. However, further investigation need be carried out to confirm its efficiency in vivo.

The protective effects of melatonin against cryopreservation-induced oxidative stress in human sperm.

Reactive oxygen species (ROS) production and lipid peroxidation during cryopreservation harm sperm membrane and as a result reduce the recovery of motile sperm. The antioxidant effects of melatonin on different cells have been widely reported. This study was aimed to evaluate changes in post-thaw motility, viability, and intracellular ROS and malondialdehyde (MDA) in response to the addition of melatonin to human sperm freezing extender. Semen of 43 fertile men was collected and each sample was divided into eight equal aliquots. An aliquot was analyzed freshly for viability, motility, and intracellular ROS and MDA. Melatonin was added to the recommended human freezing extender to yield six different final concentrations: 0.001, 0.005, 0.01, 0.05, 0.1, and 1 mM. A control group without melatonin was also included. Two weeks after cryopreservation, samples were thawed and pre-freeze analyses repeated. Obtained results showed that cryopreservation significantly (P <0.05) reduces viability and motility, but increases intracellular ROS and MDA of human sperm. The semen extender supplemented with various doses of melatonin (except for 0.001 mM) significantly (P <0.05) increased motility and viability, but decreased intracellular ROS and MDA levels of cryopreserved sperm after the thawing process, as compared with the control group. We also found that the most effective concentration of melatonin in protecting human spermatozoa from cryopreservation injuries was 0.01 mM. These findings suggest that melatonin exerts its cryoprotective effects on spermatozoa possibly by counteracting intracellular ROS, and thereby reduces MDA generation. This finally leads to increase of post-thaw viability and motility of cryopreserved spermatozoa.

Unexpectedly high occurrence of Escherichia coli and Staphylococcus aureus isolates from raw milk in Ilam, Western Iran.

Raw milk contains diverse nutritional components that provide a suitable medium for spoilage and the growth of potentially pathogenic microorganisms. Unpasteurized milk consumption by a large number of people can threaten health and increase public concerns. In this study, sixty-two raw cow’s milk samples were collected from the dairy farms of Ilam, Western Iran. All samples were collected in sterilized containers and were transferred via ice boxes to the laboratory. Isolates were then identified by standard methods. Totally, 88.7% (n=55) of samples were contaminated. Our study also showed that Escherichia coli had a high prevalence among isolates (43: 69.4%), while Klebsiella pneumoniae and Klebsiella planticola showed the lowest prevalence (1: 1.6%). Staphylococcus aureus was also detected in 17.7% (n=11) of samples. The raw milk microbial contamination is complex. Some of the microorganisms threaten public health via different traits, therefore it is recommended that raw milk consumption should be avoided.

Enterococcus faecalis as multidrug resistance strains in clinical isolates in Imam Reza Hospital in Kermanshah, Iran.

The current study aimed to investigate the prevalence of vancomycin-resistant Enterococcus in E. faecalis and E. faecium and antimicrobial susceptibility patterns, then dominant genes responsible for vancomycin resistance were determined. For this propose, 180 clinical isolates of Enterococcus were subjected for identification and antibiotic susceptibility assay. Then, the gene responsible vancomycin resistant strains were determined. The results demonstrated the E. faecalis as a dominant Enterococcus. Resistance to erythromycin was dominant and multidrug resistance strains observed in E. faecalis. vanA was responsible for vancomycin resistance. In conclusion, a high rate of resistance to antibiotics in Enterococcus is clearly problematic, and a novel strategy is needed to decrease resistance in Enterococcus.

Vitiligo: symptoms, pathogenesis and treatment.

Vitiligo is an acquired cutaneous disorder of pigmentation, with an incidence of 0.5% to 2% worldwide. There are three major hypotheses for the pathogenesis of vitiligo that are not exclusive of each other: biochemical/cytotoxic, neural and autoimmune. Recent data provide strong evidence supporting an autoimmune pathogenesis of vitiligo. As vitiligo can have a major effect on quality of life, treatment can be considered and should preferably begin early when then disease is active. Current treatment modalities are directed towards stopping progression of the disease and achieving repigmentation. Therapies include corticosteroids, topical immunomodulators, photo(chemo)therapy, surgery, combination therapies and depigmentation of normally pigmented skin. It seems that traditional Chinese medicine could be more effective than the current treatment for vitligo.

Evaluation of Chronic Hepatitis B Infection in Patients with Seronegative HbsAg.

BACKGROUND: It is estimated that about 370 million people are chronic carriers of HBV worldwide. Apparently 3% of Iranian populations are chronic carriers of this virus. We aimed to evaluate the viral DNA in biological fluids of chronic hepatitis patients compared to a control group. METHODS: The current case-control study was designed to evaluate the viral DNA in biological fluids of 70 chronic hepatitis patients compared to a control group using ELISA, PCR and Real Time. RESULTS: All individuals (100%) in case group were HBsAg positive while in control group only 2 individuals (2.8%) were HBsAg positive. Three individuals, in control group were positive using PCR and Real Time PCR indicating that about 7% of those in control group were chronic carriers of HBV. The interesting point was the copy of viral DNA; (5.49 ×10(4), 2.162×10(3) and 7.26×10(6)) for 3 chronic carriers using sera while it was about (5.71×10(3), 1.45×10(2) and 2.56×10(5)) using ear cerumen confirming the necessity of investigating for the carriers of HBV in different biological fluid and by different methods. CONCLUSION: It can be concluded chronic carriers of hepatitis B are much more than what is diagnosed by routine diagnostic tests. On the other hand ELISA alone can not be relied on as a complete test for screening of chronic carriers in hepatitis B. PCR and Real Time PCR are more reliable tests for this purpose.

Effect of tannin extract against Pseudomonas aeruginosa producing metallo beta-lactamase.

Carbapenems are the most potent beta-lactam agents with a broad-spectrum activity against Gram-negative and Gram-positive bacteria. They are stable in the presence of penicillinases and cephalosporinases. This study was focused on frequency of metallo beta- lactamase (MBL) among Pesudomonas aeruginosa strains isolated in patients with urinary tract infection, effect of tannin against PA positive strains which produced blaVIM or blaIMP and both of these genes (Species). Detection of MBL was performed by phonotypic and genotypic methods. Tannin extract was tested against P. aeruginosa producing MBL. During the study period, 240 P. aeruginosa isolates were identified. Among them 64 (26.6 percent) isolates were imipenem non-susceptible and confirmed by imipenem/EDTA. Our results revealed that the growth of blaVIM positive P. aeruginosa inhibited at 15 microg/ml concentration. The experiment repeated for blaIMP-positive P. aeruginosa and P. aeruginosa which harbored blaIMP and blaVIM, the results showed 35 microg/ml was the best concentration for inhibition of P. aeruginosa-positive blaIMP and also P. aeruginosa blaIMP and blaVIM. In conclusion, tannin was effective against P. aeruginosa producing blaVIM and blaIMP and both of them so it can be substituted with common antibiotics. The result showed significantly P. aeruginosa-harbored blaIMP was more responsible for imipenem resistance than P. aeruginosa-positive blaVIM. Interestingly, tannin was more effective against MBL-P. aeruginosa in comparison with current antibiotics.