Selected plant essential oils inhibit biofilm formation and luxS- and pfs-mediated quorum sensing by Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) causes food-borne outbreaks worldwide and the bacteria form antimicrobial-tolerant biofilm. We investigated the abilities of Thymus daenensis and Satureja hortensis essential oils (EOs) to inhibit bacterial growth, biofilm formation and quorum sensing (QS) by E. coli O157:H7. The tested EOs were isolated from plant material by hydrodistillation and analysed under chromatography-mass spectrometry. The antibacterial and anti-biofilm activities of the EOs were determined by microdilution broth and microtitre-plate (MtP) tests, respectively. The QS inhibitory (QSI) potential was examined by inhibition of swimming and swarming motility at sub-MIC levels. Real-time quantitative reverse transcription PCR (RT-qPCR) was used to determine the expression of QS-system-related genes. The MICs for T. daenensis and S. hortensis EOs against EHEC were 3·12 and 6·25 µg ml-1 , respectively and the MBCs were 6·25 and 12·5 µg ml-1 , respectively. The MtP test showed a significant (P < 0·05) inhibitory and disruptive effect on both EOs for EHEC biofilm formation at MIC/2 (1·56 µg ml-1 for T. daenensis; 3·12 µg ml-1 for S. hortensis) and MIC/4 (0·78 µg ml-1 for T. daenensis; 1·56 µg ml-1 for S. hortensis) concentrations. Gene expression analysis revealed significant down-regulation of luxS and pfs following treatment with MIC/2 concentrations. The results of the present research point to the promising antibacterial, anti-biofilm and anti-QS potential of T. daenensis and S. hortensis EOs against E. coli O157:H7.

Serotype Distribution and Antimicrobial Resistance of Salmonella Isolates in Human, Chicken, and Cattle in Iran.

Salmonellais a foodborne zoonotic enteric bacterium able to infect both humans and animals. This study aimed to identify the antimicrobial resistance of Salmonella serovars isolated from human, cattle, and poultry. Moreover, we investigated the probable transmission trends of antibiotic-resistant Salmonella isolates from food animals to human. A total of 242 Salmonella isolates collected from various human and animal sources were serotyped. The polymerase chain reaction was performed to detect the invA virulence gene. The isolates were subsequently tested against 14 antimicrobials and the resistance rates among the isolates from three sample sources were statistically analyzed by the Chi-Square test. Serotyping revealed the isolates belonged to various serovars with the dominance of Enteritidis (37%), Typhimurium (35.3%), and Infantis (21.1%). A high frequency of resistance to streptomycin was observed followed by tetracycline, trimethoprim, sulfonamides, spectinomycin, chloramphenicol, florfenicol, ampicillin, kanamycin, ceftazidime, and cefepime. In addition, multidrug resistance was observed in more than 40% of the isolates. The results of the statistical analysis showed a significant relationship (P ˂ 0.001) between the rate of antibiotic resistance among the three sources of Salmonellaisolates. Furthermore, the antibiotic resistance had a statistical relationship between the different serotypes isolated from different sources. These findings demonstrate the possible transmission of resistance to human from animal sources. The prevalence of the Typhimurium, Enteritidis, and Infantis serovars in both human and animals suggested that Salmonella contamination in chicken and cattle may be the major source of salmonellosis in human. The high incidence of antibiotic resistance in Salmonellaisolates along with the close relationship between the antimicrobial resistance of animal and human isolates indicate the role of food animal products as an important source of resistance.

Phylogenetic Analysis of bor Gene in an Escherichia coli Strain 1378 (O78:K80) Isolated from an Avian Colibacillosis Case in Tehran, Iran.

Colibacillosis is known as a fatal bacterial disease resulting in a high level of commercial loss worldwide. This study amid to elucidate the sequence, genetic characteristics, and phylogeny of the bor gene in Escherichia coli (E. coli) strain c1378 (O78:K80) isolated from avian colibacillosis in Iran and develop a rapid and optimal polymerase chain reaction (PCR) molecular-based technique with specific primers to detect this gene in E. coli. A virulent avian E. coli (i.e., laboratory designation E. coli strain c1378) isolated from a chicken with systemic colibacillosis from a broiler farm in Tehran, Iran, in 2004 was used as a source of the bor gene. After DNA extraction, PCR method was used to amplify the bor gene. A 658 bp fragment of the bor gene was amplified, sequenced, blasted, and phylogenetically studied. The most similar sequences to the bor gene in E. coli strain c1378 were E. coli APEC O78, Enterobacteria phage HK630, and Escherichia coli BW2952, respectively. There was a high similarity between the bor gene in E. coli bacteria with their phage and plasmid. Moreover, a high similarity was observed between the bor and iss genes (approximately 92%) showing that they were homologous genes. In addition, the similarity analysis of different bacterial species, as well as their plasmid and bacteriophage, to the bor gene indicated that the highest similarity to O78:K80 was related to Paracoccidioides brasiliensis, Bacillus thuringiensis CT43 plasmid pBMB0558, and Salmonella enterica subsp. enterica serovar Kentucky strain CVM29188 plasmid, respectively. Altogether, the results of the present study confirmed the presence of the bor gene in the studied isolates and clarified its sequence, phylogenetic relationship, and similarities of E. coli strain c1378 (O78:K80) isolated from avian colibacillosis.

Antibacterial and anti-PmrA activity of plant essential oils against fluoroquinolone-resistant Streptococcus pneumoniae clinical isolates.

Streptococcus pneumoniae (pneumococcus) is recognized as one of the major cause of infections in communities and hospitals. In this study, anti-pneumococcal and anti-efflux pump activity of two medicinal plants (Thymus daenensis and Origanum vulgare) essential oils were evaluated. Checkerboard assay test was performed for investigation of the effects of selected EOs on ciprofloxacin (CIP) and ethidium bromide (EtBr) uptake in pmrA-overexpressed fluoroquinolone-resistant pneumococcus. Using quantitative real-time RT-PCR the PmrA efflux pump gene (pmrA) expression was evaluated following treatment with selected EOs. Gas chromatography-mass spectrometry analysis was performed for identifying the major components of the tested EOs. The minimum inhibitory concentration (MIC) values for pneumococcus isolates were 0·625-2·5 µl ml-1 for T. daenensis and 1·25-5 µl ml-1 for O. vulgare EOs. We confirmed that in all strains T. daenensis and O. vulgare have a total or partial synergistic effects with CIP and EtBr (FICI from 0·14 to 0·75). In other hand MIC/2 concentration of T. daenensis and O. vulgare EOs caused a significant downregulation of pmrA gene (P < 0·05) in seven of eight strains. This study showed that T. daenensis and O. vulgare EOs have strong antimicrobial and anti-efflux pump activity against clinical isolates of S. pneumoniae and might be useful in controlling pneumococcal infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This study introduced Thymus daenensis and Origanum vulgare essential oil as new antibacterial and anti-efflux pump agents against fluoroquinolone-resistant Streptococcus pneumoniae clinical isolates. These findings indicate that combination of these two essential oils with fluoroquinolone antibiotics may provide alternative methods to overcome the fluoroquinolone-resistant S. pneumoniae.

Molecular identification of Salmonella Infantis isolated from backyard chickens and detection of their resistance genesby PCR.

This study aims at molecular identification of Salmonella Infantis isolated from backyard chickens and the detection of their antibiotic resistance genes. A total of 46 Salmonella-suspected samples isolated from backyard chickens of northern Iran were collected. Serotyping was done by the traditional method and then confirmed by PCR. Antimicrobial susceptibility of the isolates against 13 antimicrobial agents was determined by the standard disk diffusion method. There were 44 samples identified as Salmonella. Serotyping results showed that all 44 isolates belonged to serogroup C1 and serovar Infantis. The most resistance observed was to tetracycline and doxycycline (100%), chloramphenicol (79%) and florfenicol (72%). The floR, catI, tetA and tetG genes were used for the detection of florfenicol chloramphenicol and tetracycline resistance. In order to identify the phenotypic resistance in strains which showed resistance genes by PCR, colony PCR and culture on plates each containing antibiotic was performed simultaneously. All the Salmonella Infantis resistant to florfenicol and chloramphenicol harbored floR and catI. None of the Salmonella resistant to tetracycline carried tetA or tetG. The result of colony PCR and culture in antibiotic medium confirmed the results of PCR and indicated phenotypic resistance in these samples.

Gene disruption in Salmonella typhimurim by modified λ Red disruption system.

There are many techniques to knock out directed genes in bacteria, some of which have been described in Salmonella species. In this study, a combination of SOEing PCR method and the λ Red disruption system were used to disrupt phoP gene in wild type and standard strains of Salmonella typhimurium. Three standards PCR and one fusion PCR reactions were performed to construct a linear DNA including upstream and downstream of phoP gene and Kanamycin cassette. As a template plasmid, we used pKD4 which carries kanamycin gene flanked by FRT (FLP recognition target) sites. The resulting construct was electroporated into prepared competent cells of S. typhimurium. The transformants colonies related to the standard strain appeared on the LB-Km-agar plates after incubation, but there was no colony on LB-Km-agar plates corresponding to the wild type strain. The failure in transformation of the wild type strain may be because of inflexibility of the λ Red disruption system in this strain or its unique restriction-modification system. However, by this construct we are able to generate phoP mutant in many of the Salmonella species due to high homology of the phoP gene which exists in different species.

The production of monovalent and anti-idiotype antivenom against Mesobuthus eupeus (Scorpionida: Buthidae) venom in rabbits.

The antivenom production against poisonous creatures encounters a number of difficulties. Interestingly, according to the network theory the conventional antigens are not necessarily needed for producing antibodies against the venoms. In this investigation, the antivenom against Mesobuthus eupeus venom was produced based on the aforementioned theory. Polyclonal antibodies against M. eupeus venom were obtained from the immunized rabbits and the specific antibodies were isolated. After separation of Fab2, immunization process and production of the monovalent and anti-idiotype, these antivenoms were analyzed for the determination of their neutralizing power. The level of the produced antibodies in different stages of this study was also measured by ELISA assay. Four hundred and fifty micrograms of the venom can be neutralized by 4.2, 18 and 291 mg of monovalent, polyvalent and anti-idiotype antivenom, respectively. The ELISA results revealed that idiotypic antigens were six times more immunogenic than anti-idiotypes. The anti-idiotype antivenom can be produced on a large scale with minimum venom consumption. In addition, they are non-toxicant in immunized animals and can be used as a vaccine in people at the risk of scorpion stings.