Design and assessment of novel synthetic peptides to inhibit quorum sensing-dependent biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most common biofilm-producing bacteria, often leading to long-term and chronic infections. The LasR regulator protein acts as the central regulator of the quorum sensing (QS) system and coordinates the expression of some virulence and biofilm genes. In this study, novel peptides (WSF, FASK, YDVD) were designed for binding to the domain of the transcriptional activator of the LasR protein and interfere with LasR in the QS system of P. aeruginosa. The effects of these peptides on biofilm production, expression of biofilm-related genes (AlgC, PslA, PelA), and growth of planktonic P. aeruginosa were investigated. All three peptides inhibited the growth of P. aeruginosa planktonic cells at 1600 µg ml-1 and exhibited anti-biofilm effects at sub-inhibitory concentrations (800 µg ml-1). Measurements of the mRNA levels of biofilm-related genes at sub-inhibitory concentrations of the designed peptides showed a significant decrease.

Synthesis and Biological Properties of Silver Chloride Nanoparticles Using Cell-free Extracts of Aeromonas ‎hydrophila and Antibacterial Activity against Drug-Resistant Bacteria

Abstract Microorganisms have been studied as potential biological factories for the synthesis of silver nanoparticles. In the present study, the cell-free extract of Aeromonas ‎hydrophila was used to synthesize silver nanoparticles because the extract has a dual role in reducing and stabilizing silver nanoparticles. In this study, silver nanoparticles were synthesized using Aeromonas hydrophila. Synthetic nanoparticles were examined using ultraviolet-visible spectroscopy, X-ray diffraction, Fourier transform infrared (FT-IR), transmission electron microscopy (TEM) and X-ray diffraction (EDX) spectroscopy. In this study, antimicrobial properties of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa and anti-cancer properties (MCF-7, HepG-2) of silver nanoparticles were investigated. The synthesis of silver nanoparticles was confirmed by ultraviolet-visible spectroscopy and X-ray diffraction. TEM images detected the spherical shape of nanoparticles of various sizes in the range of 1-20 nm. FT-IR analysis demonstrated that enzyme, protein and carbohydrate compounds can be proven as stabilizing agents on the surface of silver nanoparticles. The resulting nanoparticles had strong antibacterial activity against drug-resistant bacteria. Silver chloride nanoparticles were also toxic to MCF-7 and HepG-2 cancer cells. The green synthesis method is cost-effective, environmentally friendly and an easy alternative to conventional silver nanoparticle synthesis methods.

Bayesian estimation of sensitivity and specificity of a PCR method to detect Coxiella burnetii in milk and vaginal secretions in sheep and goat samples.

Coxiella burnetii is a gram-negative and polymorphic rod bacterium that causes Q fever, a common zoonotic disease distributed worldwide. Widespread occurrences of the disease outbreaks indicate the importance of coordinated animal and human health efforts to control these outbreaks. Different tests are available to determine the C. burnetii infection status of a flock, but false negative responses may occur, as infectious animals can shed bacteria in milk intermittently, especially during an asymptomatic infection. In this study, a Bayesian latent class model was implemented to estimate the sensitivity (Se) and specificity (Sp) of a PCR method for the detection of C. burnetii in milk samples (PCRM) and vaginal swabs (PCRV) from Iranian sheep and goats. A nested PCR assay was conducted to detect infected animals among 170 milk samples and 170 vaginal swabs from goat flocks and 170 milk samples and 170 vaginal swabs from sheep flocks. We implemented a Bayesian latent class model to estimate the Se and Sp of a PCR method for the detection of C. burnetii in milk samples and vaginal swabs from sheep and goats. Estimations were based on the cross-classified results of PCRM and PCRV from the sheep and goat subpopulations. Positivity was 17.6 and 33.5%, respectively, for PCRM and PCRV samples among sheep. In goats, the apparent prevalence was 32.9 and 56.4% in PCRM and PCRV samples testing positive, respectively. This indicated the lower sensitivity of PCRM. The Se of PCRV was significantly higher than Se of PCRM, which corresponded to a higher rate of vaginal-positive, milk-negative PCR samples. In contrast, Sp of PCRV was lower than Sp of PCRM, representing the higher false-positive rate of vaginal swabs. The PCRV outperformed PCRM in terms of identifying latently infected sheep and goats; however, neither method could identify all latently infected sheep and goats, thus the combination is recommended to maximize our ability to identify infected animals. The true prevalence of C. burnetii infection was higher in Iranian goats than sheep.

Bacterial ghost of avian pathogenic E. coli (APEC) serotype O78:K80 as a homologous vaccine against avian colibacillosis.

Avian Colibacillosis is among the major causes of economic loss in the poultry industry worldwide, with a more vivid impact on developing countries. The involvement of several bacteria has made it challenging to develop effective vaccines for this disease, particularly because it is notoriously difficult to make a vaccine that contains all the contributing pathogenic bacteria. Here, we report the design and fabrication of a bacterial ghost (BG) of E. coli O78:K80, which is among the major bacterial serotypes responsible for this disease. The generated ghost is then exploited as a homologous vaccine against Avian Colibacillosis. We demonstrate that hole formation in the cell wall of E. coli O78:K80 can happen properly in optical densities as high as 0.8 compared to the 0.3-0.4 standard for bacteria like E. coli TOP10. This is especially advantageous for mass production of this ghost which is a vital factor in development of any BG-based vaccine. Compared to E. coli TOP10, we faced a great challenge in transforming the wild type bacteria with the E-lysis plasmid which was probably due to higher thickness of the cell wall in O78:K80. This, however, was addressed by treating the cell wall with a different combination of ions.The vaccine was administered to Ross 308 broiler chickens via injection as well as through their respiratory system at a dose of 1010 BGs, repeated 3 times at weekly intervals. Chickens were then challenged with the wild type O78:K80 at a dose of 1011 bacteria together with Infectious Bronchitis H120 vaccine (as immunosuppressant) one week after the last immunization. Air sac lesions were significantly reduced in BG vaccinated groups in comparison with the control group. The levels of IFNγ, IgA and IgY were measured in the serum of immunized chickens as an indication of immune response and were compared with those of the chickens vaccinated with killed bacteria. The results show that O78:K80 BG can be used as an efficient homologous vaccine against Colibacillosis disease in poultry. We expect our findings can serve as the starting point for designing more sophisticated vaccines that contain all three major pathogenic bacteria involved in avian Colibacillosis.

A high prevalence of tylosin resistance among Staphylococcus aureus strains isolated from bovine mastitis.

The macrolides appear to have considerable effects for treatment of bovine mastitis because of excellent diffusion into the mammary gland, long half-life, low protein binding, high intracellular concentration and lipid solubility. Acquired resistance to macrolides in Staphylococcus aureus is primarily related to target-site modification through acquisition of an erm gene. In the present study the prevalence of both phenotypic and genotypic tylosin resistance in S. aureus isolates (n = 103) from subclinical mastitis in nine dairy farms belonging to three different province of Iran were investigated. Overall, ermA, ermB and ermC was found in 7.80%, 32.00%, and 20.40% of S.aureus isolates, respectively. A very high percent of isolates (56.90%) were resistant to tylosin. MIC90 and MIC50 values were 64 and 32 µg mL-1, respectively. Most of tylosin resistant isolates did not harbour any erm gene but ermB was dominant gene among 58 tylosin resistant isolates of S. aureus. In overall, tylosin resistance was prevalent in S. aureus isolates obtained from bovine mastitis in Iran.

Detection of lytA, pspC, and rrgA genes in Streptococcus pneumoniae isolated from healthy children.

BACKGROUND AND OBJECTIVES: Many surface proteins are implicated in nasopharyngeal colonization and pathogenesis of Streptococcus pneumoniae. Some of these factors are candidate antigens for protein based vaccines. New vaccine designs focus on the surface proteins (e. g., pspA and pspC) and also cytolysin, and pneumolysin. In this study, 3 key virulence genes, lytA, pspC, and rrgA, which encoded surface proteins, were detected among S. pneumoniae isolates. MATERIALS AND METHODS: A total of 260 nasopharyngeal swabs were collected from healthy children under 6 years old attending day care centers in Mashhad, Iran. Isolates of S. pneumoniae were confirmed by optochin susceptibility and colony appearance and also by PCR for cpsA gene. The presence of lytA, pspC, and rrgA genes were also detected by PCR. RESULTS: A total of 59 isolates were confirmed as S. pneumoniae. Among these isolates, 50 (84.74%), 19 (32.20%), and 2 (3.38%) were positive for lytA, rrgA, and pspC genes respectively. The presence of these genes among S.pneumoniae isolates were as follows: 1) rrgA, lytA, pspC (1 isolate), 2) rrgA, lytA(17isolates), 3) pspC (2 isolate), 4) lytA (50 isolates). CONCLUSION: cpsA gene was specific for detection of S. pneumoniae isolates which were colonized in nasopharynx. The lytA gene was the most frequent gene among the S. pneumoniae isolates, and combination of rrgA, lytA was the most observed pattern. Thus, it is important for future monitoring of vaccine formulation in our country.

A comparison analysis of Listeria monocytogenes isolates recovered from chicken carcasses and human by using RAPD PCR.

Chicken meat is a valuable source of protein and consumption of it continues to rise day to day. The aim of the present study was to evaluate the genetic homogeneity of Listeria monocytogenes (L. monocytogenes) isolates obtained from chicken carcasses and human. Random Amplification of Polymorphic DNA (RAPD) PCR with three different primers were used to analyze the 31 L. monocytogenes isolates recovered from human and chicken carcasses. Primers were D8635, HLWL74, and OPM01. Scanned images of RAPD-PCR products were analyzed using Photocap software. The data were analyzed by SPSS software using Jaccard distance matrix and Ward's hierarchical cluster technique, isolates were clustered and displayed in dendrogram form. Molecular serotyping of the isolate was done. Most of the isolates were grouped into two serogroup IIb and IIa. However some of them were serotyped as IVb serogroup. In the RAPD assay, all of the primers gave amplified bands. Among these three primers, OPM01 had the most discriminatory power due to producing polymorph bands. Totally, 75 different bands with sizes ranging from 150 bp to 3300 bp, were produced. The dendrogram for Listeria monocytogenes isolates from chicken and human showed five different clusters (designed as A to E). In this study, there wasn't any association between food and human isolates of L. monocytogenes. RAPD has more discriminatory power than serotyping. On the other hand, there were different RAPD profiles among isolates of the same serotype and also, similar RAPD profile among different serotypes were observed.

Analysis and evaluation of antibacterial effects of new herbal formulas, AP-001 and AP-002, against Escherichia coli O157:H7.

AIMS: Over the last few years, there has been an increasing interest in the use of natural substances for promoting human and animal health. Antibiotic resistance, enhanced by excessive use of antibiotics, has encouraged human and animal health authorities to consider and employ alternative approaches, including herbal and botanical medicine to combat invading microorganisms. Herein, the essential oils of Thymus vulgaris, Allium cepa, Allium sativum, Eucalyptus globulus, Salvia officinalis, Dianthus caryophyllus, Mentha spicata and Mentha piperita, were evaluated for their antibacterial activities against standard Escherichia coli O157:H7 by disk diffusion method. MAIN METHODS: Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs) were assessed via microdilution assay. In the next step, two combinations of these essential oils were formulated. Their in-vitro antibacterial effects were evaluated and compared with a commercially available herbal drug containing T. vulgaris essential oil. KEY FINDINGS: According to the results, the two new formulated essential oil combinations had more potent antibacterial effects against E. coli when compared to the commercial herbal drug. SIGNIFICANCE: The presented data indicate the potential antibacterial activity of these newly formulated essential oil remedies to be employed in the poultry industry in the fight against colibacillosis, although this claim has to be examined in experimental and clinical trials.

Evaluation of different primers for detection of Brucella in human and animal serum samples by using PCR method.

BACKGROUND: The Polymerase Chain Reaction (PCR) method can overcome the limitations of conventional methodology. The aim of this study is to evaluate three primer pairs broadly used B4/B5, F4/R2 and JPF/JPR, for detection of Brucella by PCR, in human and animal serum samples and to determine analytic sensitivity of primers. METHODS: Total of 68 serum samples were collected during the acute phase of brucellosis. 10-fold serial dilutions were prepared from bacterial suspension and serum suspension using Brucella abortus S19. DNA was isolated using boiling. The best dilution from DNA was determined for PCR with three primer pairs. PCR was performed using primer pairs on all bacterial dilutions, serum dilutions, 1/200 dilutions and serum samples. Comparison of sensitivity between three primer pairs was performed with statistical analysis. RESULTS: The best DNA dilution was 1/200. From 68 serum samples, 54 cases (79.41%), 44 cases (64.70%) and 35 cases (51.47%), were positive by PCR with B4/B5, F4/R2 and JPF/JPR respectively. B4/B5, F4/R2 and JPF/JPR were able to identify 9 × 10(2), 9 and 9 × 10(5) bacteria in 1 ml of bacterial suspension and 9 × 10(4), 9 × 10(5) and 9 × 10(7) bacteria in 1 ml of dilution 1/200 of Serum dilutions respectively. The differences between primers by statistical analysis were significant for human, animal and total samples. CONCLUSION: No band was observed in dilutions one of DNA isolated from serum. Therefore, to decrease the effects of inhibitors, DNA was diluted. When DNA isolation is boiling, F4/R2 and B4/B5 have the greatest sensitivity for purified bacteria and serum in the detection of Brucella respectively. DNA isolation by boiling can decrease the PCR costs.

Genotyping of Clostridium perfringens isolated from broiler meat in northeastern of Iran.

Clostridium perfringens (C. perfringens) is an important cause of bacterial food poisoning worldwide. The disease is caused by C. perfringens enterotoxin (CPE) encoded by cpe gene. The aim of this research was to identify the different types of C. perfringens and the presence of cpe gene in isolated bacteria from broilers' meat marketed in retail meat shops of Mashhad city in Northeastern of Iran. After isolation of C. perfringens using conventional culture method and confirmation by specific 16S rDNA gene, a multiplex polymerase chain reaction assay with specific primers, were performed for toxin typing of isolates. Clostridium perfringens was isolated from 31 broilers' meat samples (15.50%) out of 200 samples and for toxin typing the results showed 9 isolates as type A (29.03%) and 22 isolates as type C (70.96%). In this study, cpe-positive C. perfringens were detected in eight isolates of type C (25.00%). Our results indicated that C. perfringens type C is the most common type in broiler chicken carcasses.

Bovine salmonellosis in northeast of Iran: frequency, genetic fingerprinting and antimicrobial resistance patterns of Salmonella spp.

OBJECTIVE: To evaluate serovar and antimicrobial resistance patterns of Salmonella spp isolated from healthy, diseased and necropsied cows and calves in this observational study. METHODS: Nineteen isolates recovered from feces and tissues of salmonellosis-affected animals of two commercial farms in north-east of Iran. In second part of the study, the two farms were sampled 4 times with an interval of 2 month. The samples included calves' feces, adult cows' feces, feeds, water, milk filters, and milk fed to calves. Five Salmonella were isolated from 332 fecal samples collected from calves and peri-parturient cows. No Salmonella was recovered from water, feed, milk filers and milk fed to calves. RESULTS: Salmonella Typhimurium was the most frequently isolate among all sero-groups. S. Dublin was only accounted for 8% (two out of 24) of isolates. Isolated Salmonella strains were used for the ERIC PCR DNA fingerprinting assay. Our results grouped Salmonella isolates into 3 clusters, suggesting that specific genotypes were responsible for each sero-group of Salmonella. The results also revealed diversity among Salmonella isolates in cluster III (sero-group B). Eighteen out of 19 Salmonella spp. were resistant to oxytetracycline. Five isolates out of 19 showed more than one drug resistance. Multi-drug resistance was seen only among Salmonella Typhimurium isolates. Enrofloxacin was the most susceptible antibiotic against all isolates in this study. CONCLUSION: The emergence of multiple antibiotic-resistant strains of Salmonella Typhimurium should be of great concern to the public. No correlation between ERIC fingerprinting and resistance patterns of Salmonella isolates was found, which indicates resistance to antimicrobial agents was not related to specific genetic background.

Genotyping of Clostridium perfringens isolated from healthy and diseased ostriches (Struthio camelus).

BACKGROUND AND OBJECTIVES: Clostridium perfringens is more prevalent type of clostridia genus isolated from the intestinal tract of ostrich (Struthio camelus). Necrotic enteritis (NE) is a potentially fatal gastrointestinal (GI) disease of poultry and other avian species, which produces marked destruction of intestinal lining in digestive tract caused by C. perfringens. Pathogenicity and lesions are correlated with the toxins produced, thus toxin typing of the bacterium has diagnostic and epidemiological significance. The aims of the present study were to determine the biotypes of C. perfringens among ostrich's farms either diseased and healthy ones and to screen the isolates for major toxin genes (cpa, cpb, etx, and iA, cpb2, and cpe). MATERIALS AND METHODS: Thirty isolates of C. perfringens were obtained from NE-positive and NE-negative ostrich flocks in Khorasan-e-Razavi porvince and analyzed by multiplex PCR assay. RESULTS: All isolates were positive for alpha toxin gene (cpa) and five of those were positive for beta toxin gene (cpb). The presence of cpb2 gene was detected in a high percentage of isolates originating from both healthy (93.3%) and diseased flocks (80%). None of the isolate carried enterotoxin gene (cpe). CONCLUSION: The results suggest that types A and C of C. perfringens are the most prevalent types in ostrich in Iran. Due to detection of beta2 toxin gene in isolates from both healthy and diseased birds, it appears that the presence of cpb2 is not considered a risk by itself.

Length of dsRNA (poly I:C) drives distinct innate immune responses, depending on the cell type.

Poly I:C, a synthetic dsRNA analogue, has been used extensively for decades to study innate responses in vivo and in different cell types. We have found substantial variability while using poly I:C from different sources. In this study we found that poly I:C from 2 commercial sources induced sharply opposite responses in myeloid and fibroblasts, depending on the length of the poly I:C. Although short poly I:C (≈ 1-1.5 kb) induced greater amounts of TNF-α, IL-8, and IFN-ß and a stronger antiviral response in myeloid cells, it was a poor inducer in fibroblasts. By contrast, long poly I:C (>5 kb) preferentially elicited higher cytokine and antiviral responses in fibroblasts and showed diminished responses in myeloid cells. Poly I:C activated NF-κB and STAT-1 signaling in a length- and cell-type-dependent fashion. Mechanistically, short poly I:C was better internalized in the myeloid cells and long poly I:C in the fibroblasts. Finally, long poly I:C required SR-A, whereas short poly I:C required RIG-I and Raftlin. We provide evidence that the length of dsRNA drives distinct innate responses in different cell lineages. These findings may augment in selecting the appropriate poly I:C type to design cell-type-specific potent adjuvants for vaccines against infectious diseases or cancers.

Survey of dermatological conditions in a population of domestic dogs in Mashhad, northeast of Iran (2007-2011).

university small animal clinic, 1299 Iranian domestic dogs presented from September 2007 through March 2011 to the Ferdowsi University of Mashhad Veterinary Teaching Hospital, were examined. Dermatological disorders were noted in 17.00% (221/1299) of all the dogs examined. Pruritus was the most common presenting sign, accounting for 25.35% of the dermatological consultations. It was followed by erythema, maculo-papular-pustular eruptions (16.97%), erosive or ulcerative lesions (16.74%), scaling or crusting (13.02%), alopecia (8.84%) and visible ectoparasites (7.44%). The most common primary final diagnoses were superficial pyoderma, cutaneous manifestations of canine leishmaniasis, flea infestation and allergy, tick infestation, atopic dermatitis, scabies, unspecified dermatoses, otitis, furunculosis and food allergy. There were no apparent age or sex predilections for dermatological disease as a whole. Spitz (odds ratio = 3.38; p = 0.001), Terriers (odds ratio = 2.52; p < 0.001) and German Shepherds (odds ratio = 1.90; p = 0.001) appeared to be at increased risk for dermatological disease. In addition, Khorasani large cross breed dogs (odds ratio = 0.36; p = 0.003) and mixed breed dogs (odds ratio = 0.33; p < 0.001) showed decreased risk for dermatological conditions. To the best of our knowledge, this is the first survey study on canine dermatological conditions carried out in Iran.