Antibacterial and anti-biofilm properties of carvacrol alone and in combination with cefixime against Escherichia coli.

BACKGROUND: Plant-derived compounds can be used as antimicrobial agents in medicines and as food preservatives. These compounds can be applied along with other antimicrobial agents to strengthen the effect and/or reduce the required treatment dose. RESULTS: In the present study, the antibacterial, anti-biofilm and quorum sensing inhibitory activity of carvacrol alone and in combination with the antibiotic cefixime against Escherichia coli was investigated. The MIC and MBC values for carvacrol were 250 µg/mL. In the checkerboard test, carvacrol showed a synergistic interaction with cefixime against E. coli (FIC index = 0.5). Carvacrol and cefixime significantly inhibited biofilm formation at MIC/2 (125 and 62.5 µg/mL), MIC/4 (62.5 and 31.25 µg/mL) and MIC/8 (31.25 and 15.625 µg/mL) for carvacrol and cefixime, respectively. The antibacterial and anti-biofilm potential effect of carvacrol confirmed by the scanning electron microscopy. Real-time quantitative reverse transcription PCR revealed significant down-regulation of the luxS and pfs genes following treatment with a MIC/2 (125 µg/mL) concentration of carvacrol alone and of only pfs gene following treatment with MIC/2 of carvacrol in combination with MIC/2 of cefixime (p < 0.05). CONCLUSIONS: Because of the significant antibacterial and anti-biofilm activity of carvacrol, the present study examines this agent as an antibacterial drug of natural origin. The results indicate that in this study the best antibacterial and anti-biofilm properties are for the combined use of cefixime and carvacrol.

Construction of live-attenuated Trueperella pyogenes by antibiotic treatment and sequential passage: methods for vaccine development.

Trueperella pyogenes (T. pyogenes) is a zoonotic pathogen that is cause a variety of pyogenic diseases in animals. The complex pathogenicity and various virulence factors are important challenges to produce an effective vaccine. According to previous trials, inactivated whole-cell bacteria or recombinant vaccines were unsuccessful in preventing disease. Thus, this study aims to introduce a new vaccine candidate based on a live-attenuated platform. For this purpose, first T. pyogenes was subjected to sequential passage (SP) and antibiotic treatment (AT) to lose their pathogenicity. Second, Plo and fimA expressions as virulence genes were evaluated by qPCR and then mice were challenged with bacteria from SP and AT culture by intraperitoneal route. Compared to the control group (T. pyogenes-wild type), plo and fimA gene expressions were downregulated and vaccinated mice have a normal spleen appearance in contrast to the control group. In addition, there was no significant difference between bacterial count from spleen, liver, heart and peritoneal fluid in vaccinated mice and the control group. In conclusion, this study introduces a new T. pyogenes vaccine candidate based on a live-attenuated strategy that mimics natural infection without pathogenicity for further investigation on vaccines against T. pyogenes infections.

Genomic characterisation, detection of genes encoding virulence factors and evaluation of antibiotic resistance of Trueperella pyogenes isolated from cattle with clinical metritis.

Trueperella pyogenes is one of the most important microorganisms causing metritis in post-partum cattle. Co-infection with other bacterial species such as Escherichia coli or Fusobacterium necrofurom increases the severity of the disease and the persistence of bacteria in utero. The aim of this study was to investigate the frequency of T. pyogenes strains, and their virulence and antimicrobial resistant profiles in metritis cases. The study was carried out on 200 samples obtained from metritis discharges of postpartum cattle on 18 farms around Tehran, Iran. Sixty-five T. pyogenes isolates (32.5%) were identified, of which 16 isolates were detected as pure cultures and the other 49 isolates from cultures most commonly mixed with E. coli or F. necrofurom. In terms of diversity in biochemical characteristic of T. pyogenes strains, 8 different biotypes were identified among the isolates. Single or multi antimicrobial resistance was observed in 48 isolates (73.9%), which was mostly against trimethoprim sulfamethoxazole, azithromycin, erythromycin and streptomycin. The tetracycline resistance gene tetW and macrolide resistance genes ermB and ermX were detected in 30, 18 and 25 isolates, respectively. In the screening of genes encoding virulence factors, fimA and plo genes were identified in all tested isolates. Genes encoding nanP, nanH, fimC, fimG, fimE and cbpA were detected in 50, 54, 45, 40, 50 and 37 of isolates, respectively. Thirteen different genotypes were observed in these T. pyogenes isolates. A significant association between clonal types and virulence factor genes, biochemical profile, CAMP test result, severity of the disease and sampling time was detected.

Genetic diversity and virulence genes of Salmonella enterica subspecies enterica serotype Enteritidis isolated from meats and eggs.

Salmonella enterica subspecies enterica serotype Enteritidis (S. Enteritidis) is one of the leading causes of food-borne gastroenteritis associated with the consumption of contaminated food products of animal origin. Little is known about the genetic diversity and virulence content of S. Enteritidis isolated from poultry meats and eggs in Iran. A total of 34 S. Enteritidis strains were collected from different food sources of animal origin in Tehran from May 2015 to July 2016. All of the S. Enteritidis strains were serotyped, antimicrobial susceptibility tested, and characterized for virulence genes. Pulsed-field gel electrophoresis (PFGE) was also applied for comparison of genetic relatedness. All of the strains harbored invA, hilA, ssrA, sefA, spvC, and sipA genes. A high prevalence of resistance against certain antibiotics such as cefuroxime (79.4%), nalidixic acid (47%), and ciprofloxacin (44.2%) was also observed. Regarding PFGE, S. Enteritidis strains from different sources showed considerable overlap, suggesting the lack of diversity among these isolates. Moreover, no correlation between virulence profiles or antibiotypes and PFGE clusters was observed. In conclusion, our study provided valuable information on virulence gene content, antibiotic resistance, and genetic diversity of S. Enteritidis isolated from food sources.

The evaluation of normal ocular parameters in two breeds of hedgehogs.

OBJECTIVES: The purpose of this study was to evaluate conjunctival microflora and measure normal tear production and intraocular pressure (IOP) in two breeds of hedgehogs (long-eared hedgehogs and Brandt's hedgehogs). METHODS: Forty-eight hedgehogs from two different breeds were chosen for the study. Tear production was measured using the Schirmer tear test (STT) and phenol red thread test (PRTT) in both eyes. IOP was measured using a rebound tonometer. To perform microbiological sampling one drop of tetracaine was instilled in the eyes. Two sterile microswabs were used to collect samples for the microbial and fungal culture. All the microswab samples were transferred in phosphate-buffered saline (PBS) to the laboratory for culture. Two MacConkey and two blood agar media plates were employed for each eye. Oneplate of sabouraud dextrose agar (SDA) was used for the fungal culture for each eye. Standard biochemical tests were performed to identify the isolated organisms. RESULTS: The mean STT and PRTT values were 1.6 ± 0.1 mm/min and 2.4 ± 0.3 mm/15 s in long-eared hedgehogs and 2.2 ± 0.1 mm/min and 2.5 ± 0.3 mm/15 s in Brandt's hedgehogs, respectively. Mean (SD) Intraocular pressure of right eyes in long-eared hedgehog and Brandt hedgehog were 19.7 ± 1.4 mmHg and 19.2 ± 2.4 mmHg, respectively. In the left eyes of long-eared hedgehog and Brandt hedgehog mean (SD) IOP were 19.8 ± 1.5 mmHg and 19.5 ± 2.1 mmHg, respectively. In long-eared hedgehogs, the most common bacteria were Staphylococcus epidermidis and Bacillus spp. In Brandt's hedgehogs, 24 out of 48 eyes had Staphylococcus epidermidis, which was the most commonly isolated bacterial species. CONCLUSIONS: This study established reference intervals for IOP, STT and PRTT in hedgehogs and recognised and compared ocular conjunctival microflora in two breeds of hedgehogs.

Antimicrobial and anti-biofilm effects of carotenoid pigment extracted from Rhodotorula glutinis strain on food-borne bacteria.

Background and Objectives: Carotenoid pigments are among the most important pigments and have many applications in various food, cosmetics, hygiene industries and biotechnology. These pigments are produced by plants and microorganisms including Rhodotorula spp. This research intended to study the antimicrobial and antibiofilm effects of the carotenoid pigment from Rhodotorula glutinis on food spoilage bacteria (Staphylococcus aureus and Salmonella Typhimurium). Materials and Methods: The R. glutinis was isolated from milk samples of cows with mastitis and ITS sequence-based typing was performed on them. After extracting the pigment from R. glutinis, its purity was examined using thin-layer chromatography. Following that, the broth microdilution method was used to evaluate antimicrobial effects of the pigment and MtP assay and subsequently scanning electron microscopy were used to assess the antibiofilm effects. In addition, the sub-MIC effects of the pigment on expression of quorum-sensing (QS) genes in S. Typhimurium isolates (sdiA and luxS) and S. aureus isolates (hld) were studied. Finally, the degree of toxicity of the pigment was analyzed using the MTT assay. Results: ITS sequence analysis of R. glutinis revealed that the recently separated isolates exhibited strong differences with the strains recorded in NCBI database in genetic structure. The pigment produced by R. glutinis had strong antimicrobial effects and its mean MIC against S. Typhimurium isolates (17.0 µl.ml-1) was higher than the mean MIC against the S. aureus isolates (4.1 µl.ml-1). Electron microscope images and real-time observations indicated that the sub-MIC values of the pigment suppressed biofilm formation by suppressing expression of QS genes. In addition, the mentioned pigment at high MIC concentrations did not have toxic effects on Vero cells. Conclusion: This research suggests that R. glutinis pigment is effective in destroying the planktonic form of food spoilage bacteria and degrading food spoilage biofilm-forming bacteria. Moreover, considering the low toxicity level of R. glutinis pigment for eukaryotic cells, we can suggest its use as a natural antibacterial preservative in various food materials.

Biofilm formation, antimicrobial resistance genes, and genetic diversity of Salmonella enterica subspecies enterica serotype Enteritidis isolated from food and animal sources in Iran.

OBJECTIVES: Salmonella enterica serovar Entritidis is an important pathogen in foodborne diseases and causes gastroenteritis. Several studies have investigated the genetic diversity of the strains of this bacterium. However, our knowledge of the discriminatory power of the molecular methods is limited. METHODS: In total, 34 strains of S. enteritidis were isolated from food related to animals. Antibiotic resistance of the strains, antibiotic resistance genes, and biofilm formation capacity of the strains were evaluated. For the genetic analysis of the strains, PFGE was performed using AvrII restriction enzyme. RESULTS: Among the tested antibiotics, cefuroxime, nalidixic acid, and ciprofloxacin showed the highest resistance rates (79.4%, 47%, and 44.2%, respectively). Only three antibiotic-resistance genes were identified in these strains (blaTEM: 67.6%, tetA: 9%, and sul2: 3%). In total, 91% of the strains were biofilm producers. Clustering of strains using AvrII for 26 samples with the same XbaI PFGE profile showed that these strains were in one clone and had high homogeneity. CONCLUSIONS: In conclusion, it is better to use a combination of several typing methods for typing strains that are genetically very close so that the results are reliable.

Prevalence of main quinolones and carbapenems resistance genes in clinical and veterinary Escherichia coli strains.

Background and Objectives: Antibiotics-resistant Escherichia coli strains are considered one of the most important causes of human and animal infections worldwide. The aim of current study was to detect common resistance (carbapenems and quinolones) genes by PCR. Materials and Methods: A total of 100 E. coli strains isolated from human urinary tract infection and 20 isolated strains of aborted sheep embryos were collected. PCR was performed using specific primers to detect the resistance genes. Results: Overall, among the quinolones resistance genes, qnrS resistance gene had the highest frequency (48%) and among carbapenem resistance genes, imp resistance gene had the highest frequency (45%). The frequency of resistance genes, IMP (28.45%), KPC (9.5%), VIM (9.15%), NDM (7.20%) were observed in clinical and veterinary strains, respectively. According to the results, 38.6% of E. coli strains had at least one from five genes of resistance to quinolones. The lowest frequency of resistance gene was related to qnrA, which was observed in only 29 (24.2%) strains. Conclusion: Monitoring of carbapenem and quinolone resistance in pathogenic E. coli to humans and animals has an important value in revising treatment guidelines and the national public health, and plays an important role in preventing the spread of resistant strains.

Passive immunization with anti- chimeric protein PilQ/PilA -DSL region IgY does not protect against mortality associated with Pseudomonas aeruginosa sepsis in a rabbit model.

BACKGROUND: Pseudomonas aeruginosa sepsis is associated with unacceptably high mortality and, for many of those who survive, long-term morbidity. The aims of this study were to production of IgY against chimeric protein pilQ-pilA-DSL region and killed- whole cell Pseudomonas aeruginosa O1 (PAO1) strain and their efficacy for immunoprophylaxis of sepsis caused by P. aeruginosa in a rabbit model. METHODS: Specific IgY was obtained by immunization of hens. The purity of IgY was determined by SDS-PAGE analysis. The effect of IgY on growth and hydrophobicity of P. aeruginosa were performed through time-kill assay and microbial adhesion to hydrocarbons test (MATH), respectively. The efficacy of specific IgYs was examined against P. aeruginosa sepsis in a rabbit model. The rabbits were monitored for 72 h to record physiological characters and survival. Hematologic factors, C-reactive protein, pro-inflammatory cytokines, and bacterial count from blood and solid organs were measured, periodically. RESULTS: We found that the growth inhibitory effect of the anti- killed whole cell IgY was higher than anti-pilQ-pilA IgY (P < 0.001). The hydrophobicity effect of PAO1 increased when bacteria were opsonized by anti- killed whole cell IgY while the hydrophobicity activity was decreased following incubation of PAO1 with anti-pilQ-pilA IgY in a broth medium (P < 0.001). Following intravenous (IV) administration of produced IgYs, no significant difference was observed in the survival, decrease in inflammatory mediators and clinical symptoms between the groups 48h post infection (P > 0.05). Moreover, no considerable decrease was observed in the bacterial load of blood, lungs and kidneys in rabbits treated with specific IgYs and control groups (P > 0.05). No bacteria were found in the spleen and liver samples from infected rabbits. CONCLUSION: Although produced IgYs had a good immunoreactivity, IV immunization of IgYs was not protective against P. aeruginosa sepsis in the rabbit model. Further studies are needed to assess the immune response and decreasing mortality rate using the rabbit sepsis model.

Antimicrobial resistance patterns, virulence gene profiles, and genetic diversity of Salmonella enterica serotype Enteritidis isolated from patients with gastroenteritis in various Iranian cities.

OBJECTIVES: This study aimed to evaluate antibiotic resistance profiles and presence of virulence genes among Salmonella enterica serovar Enteritidis (S. Enteritidis) isolated from patients with gastroenteritis in various regions of Iran. Moreover, genetic relatedness among the strains was assessed by pulsed-field gel electrophoresis (PFGE). MATERIALS AND METHODS: From April through September 2017, 59 Salmonella strains were isolated from 2116 stool samples. Of these strains, 27 S. Enteritidis were recovered. These strains were subjected to disk diffusion tests, polymerase chain reaction (PCR) for detection of virulence genes (invA, hilA, pefA, rck, stn, ssrA, ssaR, sefA, spvC, sipA, sipC, sopB, sopE, and sopE2), and PFGE. RESULTS: High prevalence of resistance towards cefuroxime (n = 20, 74.1%) and ciprofloxacin (n = 13, 48.2%) were demonstrated. All tested strains possessed invA, hilA, sefA, sipA, sopB, and sopE. The least prevalent virulence gene was rck (n = 6; 22.2%). Based on combinations of virulence genes, 12 virulotypes were observed. The most common virulotype was VP2 (n = 12; 44.4%), harboring all of the virulence genes except for rck. PFGE typing showed only two distinct fingerprints among tested strains. Each fingerprint had completely different virulotypes. Notably, VP4 (harboring all genes except for rck and spvC) was only presented in pulsotype A, while VP2 was confined to pulsotype B. CONCLUSION: S. Enteritidis strains were derived from a limited number of clones, suggesting that it is highly homogenous. Future works should consider combinations of other genotyping methods together with larger sample sizes from more diverse sources.

Investigation of antimicrobial susceptibility and virulence factor genes in Trueperella pyogenes isolated from clinical mastitis cases of dairy cows.

Trueperella pyogenes is an opportunistic pathogen causing important diseases including mastitis and metritis in domestic animals such as dairy cows leading to prominent economic losses in food production industry. The aim of this study was to investigate bacterial species, antimicrobial susceptibility, and presence of virulence factor genes and genotyping of T. pyogenes isolates associated with summer mastitis cases from 22 different farms around Tehran, Iran. Fifty-five percent of dairy cows with clinical mastitis symptoms was infected by T. pyogenesis indicated that this pathogen is the most important contributor to clinical mastitis in dairy cows in the present study. A significant correlation was illustrated between presence of virulence factor genes of isolated pathogen, biochemical patterns, and the utter infected types. Multidrug resistance susceptibility observed between isolates indicated the important need for prudent use of antimicrobials in treatment of mastitis caused by T. pyogenes and increased concerning of consumer health associated with recent problems of antimicrobial resistance. The categorization of isolates was implemented into seven different clonal related types by COX-PCR at 80% of similarity cutoff with significance relationship to clonal types, CAMP test result and sampling time and biochemical profile. Regarding to the results obtained at the present study, T. pyogenes can be considered as an important typically cause of purulent and acute form of clinical bovine mastitis and loss of dairy productivity. Further studies with more sample size and high-throughput omic methods in various sampling time and areas are suggested for study of this pathogen precisely.

Complete genome sequence of Trueperella pyogenes strain Arash114, isolated from the uterus of a water buffalo (Bubalus bubalis) in Iran.

OBJECTIVE: Trueperella pyogenes has been considered a major causative agent of metritis, abortion, and death in a broad range of domestic and wild animals, including cattle, swine, sheep, goats, camels, buffalo, deer, antelopes, reptiles, and birds. DATA DESCRIPTION: Here, we report the complete chromosome sequence of Trueperella pyogenes strain Arash114, isolated from the uterus of a water buffalo (Bubalus bubalis) died due to the infection caused by this pathogen. The genome assembly comprised 2,338,282 bp, with a 59.5% GC content. Annotation of the genome showed 46 tRNA genes, 6 rRNA, 1 CRISPR and 2059 coding sequences. Also, several genes coding for antimicrobial resistance such as tetW and virulence factor including plo, nanH, nanP, cbp and 4 fimbrial proteins were found. This study will advance our knowledge regarding the metabolism, virulence factors, antibiotic resistance and evolution of Arash114 strain and serve as an appropriate template for future researches.

Anti-quorum sensing effects of licochalcone A and epigallocatechin-3-gallate against Salmonella Typhimurium isolates from poultry sources.

Quorum sensing (QS) is a cell density-dependent mechanism used by many pathogenic bacteria for regulating virulence gene expression. Inhibition or interruption of QS by medicinal plant remedies has been suggested as a new strategy for fighting against antibiotic-resistant bacteria. This study aimed to assess the impact of sub-inhibitory concentrations of licochalcone A (LAA) and epigallocatechin-3-gallate (EGCG) as natural plant products on the QS-associated genes (sdiA and luxS) expression. The PCR test was used to confirm the presence of sdiA and luxS genes in 23 S. Typhimurium isolates from poultry. The quantitative real-time PCR assay was used to analyze the expression of sdiA and luxS in S. Typhimurium isolates in response to the treatment with sub-inhibitory concentrations of LAA and EGCG at 45-min time point. All S. Typhimurium isolates showed the presence of sdiA and luxS genes (100%). As result, the expression of QS-related genes was significantly reduced in S. Typhimurium isolates following treatment with LAA and EGCG. In conclusion, LAA and EGCG showed anti-QS activity with down-regulation of both sdiA and luxS genes in S. Typhimurium, suggesting potential therapeutic use of them against salmonellosis. However, it must be pointed out that the safety and efficiency of these compounds need more thorough research.

The effect of capsulated and noncapsulated egg-yolk-specific antibody to reduce colonization in the intestine of Salmonella enterica ssp. enterica serovar Infantis-challenged broiler chickens.

The antibacterial properties of egg yolk antibodies have been known for many years. Enhanced antibiotic resistance has resulted in increased need for using these antibodies as an alternative. In the present study, generation, capsulation, and inhibition growth properties of IgY directed against Salmonella enterica subsp. enterica serovar Infantis (SI) were evaluated. White Leghorn layer hens were immunized using whole cell of inactivated SI. Salmonella Infantis-specific antibody activities in sera and egg yolk were determined by ELISA. A total of 480 one-day-old male "Cobb 500" chicks were randomly divided into 8 groups, with 6 replications of 10 birds kept for 21 D. All birds from 7 challenged groups were orally inoculated with 1 mL of SI suspension (1 × 107 CFU/mL) at 3 and 4 D of age. Two groups were dietary supplemented with 5 g/kg immune powdered yolk or nonimmune powdered yolk. One group was dietary supplemented with 12.8 g/kg capsulated immune yolk (CIY). Two groups were given 8.3 mL/L of immune water-soluble yolk or nonimmune water-soluble yolk fraction in drinking water. In the antibiotic group, 1 mL/L Enrofloxacin 10% was added to drinking water. All supplements except for the antibiotic (on Day 4 for 10 D) were added on day one and continued during the experiment. Negative and positive control groups received no supplements. During the experiment, among the challenged groups, the minimum SI cecal colonization and the lowest isolation of SI from the liver (P < 0.01) was observed in the antibiotic group. Following antibiotic group, in the group receiving CIY, colonization of bacteria in ceca and liver was significantly reduced during the second and third weeks of the experiment (P < 0.01). According to the results, capsulated specific IgY has a beneficial effect in reducing the colonization of Salmonella under the conditions of this study in comparison with other forms of IgY antibody.

Prevalence and characterization of multidrug resistance and variant Salmonella genomic island 1 in Salmonella isolates from cattle, poultry and humans in Iran.

Salmonella enterica is a common food-borne pathogen with occasional multidrug resistance (MDR). Salmonella genomic island (SGI1) is a horizontally transmissible genomic island, containing an MDR gene cluster. All Salmonella serotypes are public health concern, although there is an additional concern associated with those that harbour SGI1. In Iran, there are no data on the presence of SGI1 variants in Salmonella isolates. The present study was conducted to identify MDR- and SGI1-carrying Salmonella strains isolated from various sources and to compare their genetic relatedness between human and animal sources. In total, 242 Salmonella isolates collected from chicken, cattle, and humans from 2008 through 2014 were studied. The isolates were tested for resistance to 14 antimicrobials via the disc diffusion method. They were also tested for the presence of SGI1 variants via PCR, and genetic relatedness was evaluated based on pulsed-field gel electrophoresis (PFGE). Resistance to at least one antimicrobial agent was observed in 132 (54%) Salmonella isolates (n = 242), while more than 40% of the isolates showed MDR. Based on PCR analysis, eight variants of SGI1, including SGI1, SGI1-B, SGI1-C, SGI1-D, SGI1-F, SGI1-I, SGI1-J and SGI1-O, were found in both human and animal isolates. Statistical analysis revealed no significant difference in the prevalence of SGI1 variants between human and animal isolates (p > 0.05). Macrorestriction PFGE analysis of the isolates with the same SGI1 variant and resistance patterns revealed genetic relatedness ranging from 70% to 100% among human and animal isolates. According to our review, this is the first documentation of SGI1 in Salmonella isolates in Iran. The presence of similar SGI1 variants in both humans and animals, along with their related PFGE patterns, suggests that food-producing animals may be a source of MDR Salmonella isolates in Iran.

Induction of specific cell-mediated immune responses and protection in BALB/c mice by vaccination with outer membrane vesicles from a Brucella melitensis human isolate.

Brucellosis is a worldwide bacterial zoonosis caused by Brucella spp. No approved vaccine is available for human use against the disease. In this study, outer membrane vesicles (OMVs) from a Brucella melitensis biovar 1 human isolate obtained in Iran were used to immunize BALB/c mice (n = 12) by 2 intramuscular injections with a 2-week interval. Another group of 12 mice was used as non-vaccinated controls. Two weeks after the last vaccination, six mice of each group were sacrificed, and proliferation and interferon gamma (IFNγ) production responses of their splenocytes were evaluated following in vitro stimulation with killed Brucella cells. The other mice were challenged with the virulent B. melitensis isolate. Two weeks later, mice were killed and spleens were cultured to determine the number of the challenge strain. The results showed proliferative response and IFNγ production of splenocytes from vaccinated mice (stimulation index: 2.18 ± 0.57, and 1519.35 ± 10.70 pg/mL, respectively) were significantly higher than those of control mice (stimulation index: 1.02 ± 0.02, and 210.01 ± 17.58 pg/mL, respectively). Numbers of the challenge strain in spleens of vaccinated mice were also significantly less than those in the controls with 1.6 units of protection. Our study revealed vaccination with OMVs of the B. melitensis isolate could induce specific immune responses and protection against infection in the mouse model suggesting their potential application for active immunization against brucellosis.

Genetic characterisation of methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in pets and veterinary personnel in Iran: new insights into emerging methicillin-resistant S. pseudintermedius (MRSP).

OBJECTIVES: Methicillin-resistant staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), pose a threat to animal and human health worldwide. Veterinary staff and pets may play a role in the spread of resistant clones. METHODS: A total of 125 samples from veterinary staff (n=50), dogs (n=49) and cats (n=26) were investigated. Obtained isolates were tested for the methicillin resistance gene mecA and were subjected to multiplex PCR to differentiate coagulase-positive species. Following SCCmec and spa typing, isolates were tested for the presence of various toxin and virulence genes and phenotypic resistance to common antimicrobials. RESULTS: Overall, 4 MRSA were isolated from two veterinarians and two dogs and 19 MRSP were found in eleven dogs (12 isolates) and five cats (7 isolates). The MRSA isolates possessed sea (2) and eta (3) virulence genes and the MRSP isolates possessed sea (6), expA (15), expB (1) and siet (19) genes. SCCmec type II and three spa types (t186, t1816 and t10897) were identified in the MRSA isolates. Most of the MRSP isolates belonged to SCCmec types II (2 isolates) and V (10 isolates); however, the remaining 7 isolates were untypeable and contained class C1 mec. The majority of isolates were multidrug-resistant (MDR). CONCLUSION: These findings show that pets and veterinarians could be potential sources of MDR-MRSA and MDR-MRSP in Iran. Taken together, these findings warrant future investigations on the epidemiology and public-health significance of MDR-MRSA and MDR-MRSP both in veterinarians and companion animals in Iran.

New Brucella abortus S19 Mutant to Improve Distinction Between Infected and Vaccinated Animals.

BACKGROUND: Using Brucella abortus Strain 19 (S19) to control bovine brucellosis is restricted due to induce antibodies to the O-side chain of the smooth lipopolysaccharide (LPS) which may be difficult to differentiate vaccinated and infected animals. Furthermore, it is virulent for humans and can induce abortion to cattle. OBJECTIVES: The aim of this study was to employ gene knockout B. abortus S19 for the first time to eliminate diagnostic defects and obtain the attenuated mutant strain. MATERIAL AND METHODS: The wbkA gene, which is one of the LPS O-chain coding genes, was knocked out in vaccinal Brucella abortus S19. The proliferative response and immunoglobulin M production were analyzed in wbkA deletion strain-infected BALB/c mice. RESULTS: The loss of wbkA gene function resulted in induction of the splenocyte proliferative response in mice infected by the mutant S19 strain compare to those induced by parental S19 and RB51 strains. Moreover, wbkA mutant did not induce any IgM antibody response using the enzyme-linked immunosorbent assay. CONCLUSIONS: As a result, the new mutant S19 strain had deficiency in its LPS O-chain structure, besides cannot induce IgM response then, reduce mistakes to discriminate between vaccinated and infected animal, and also can be considered as a new vaccine candidate.

ECOR phylotyping and determination of virulence genes in Escherichia coli isolates from pathological conditions of broiler chickens in poultry slaughter-houses of southeast of Iran.

Avian pathogenic Escherichia coli (APEC) are responsible for wide ranges of extra-intestinal diseases in poultry including colibacillosis, cellulitis, coligranuloma and yolk sac infection. Numbers of virulence are considered important in the pathogenicity of these diseases. The aims of the present study were phylogenetic typing and virulence genes detection in Escherichia coli isolates from colibacillosis and cellulitis of broiler chickens in poultry slaughterhouses of Shahrbabak region, Kerman, Iran. A total number of eighty three E. coli isolates were taken from broiler chickens with colibacillosis and thirty four isolates were taken from carcasses with cellulitis in the industrial slaughterhouses. Biochemically confirmed E. coli isolates were subjected to polymerase chain reaction assay to determine phylogenetic groups and presence of pap C, sfa/focDE, iucD, afaIB-C, hlyA, fimH and crl virulence genes. Colibacillosis isolates were belonged to A (54.21%), B1 (7.22%), B2 (6.03%) and D (32.53%) phylogroups. Whereas, the isolates from cellulitis cases were belonged to three main phylogroups; A (55.88%), B1 (5.88%) and D (38.24%). Statistical analysis showed a specific association between the presence of crl virulence gene and phylogroups of A and D in colibacillosis isolates. The results showed that the isolates from both diseases in broiler chickens could be assigned to various phylogenetic groups (mainly A(. Also, the virulence genes profile of cellulitis E. coli is completely different from that of colibacillosis in this region.

Antimicrobial resistance, virulence genes and genetic relatedness of Salmonella enterica serotype Enteritidis isolates recovered from human gastroenteritis in Tehran, Iran.

OBJECTIVES: Salmonella enterica serotype Enteritidis is a major serotype associated with human salmonellosis. The main objective of this study was to determine the antibiotic susceptibility patterns and the presence of virulence-associated genes among S. Enteritidis strains isolated from patients with gastroenteritis in Tehran, Iran. METHODS: Over a period of 14 months (May 2015 to July 2016), 44 S. Enteritidis isolates recovered from clinical sources were characterised for antimicrobial susceptibility and virulence genes. Possible genetic relatedness among the strains was also assessed using pulsed-field gel electrophoresis (PFGE). RESULTS: Salmonella Enteritidis isolates showed high rates of resistance to ciprofloxacin (90.9%) and nalidixic acid (77.3%). Of the 44 S. Enteritidis isolates, 30 (68.2%) were resistant to three or more antibiotics. Twenty-two different antimicrobial resistance patterns were detected among the isolates. The most frequent resistance type was antibiotype 14 (resistance to ciprofloxacin, cefuroxime and nalidixic acid), occurring in 8 (18.2%) of the isolates. Notably, all of the isolates carried invA, sefA, sipA and sopE2 virulence genes. Furthermore, 17 virulence profiles were observed among the strains. The most common virulence profile was VP1 (n=17; 38.6%), harbouring all of the virulence genes. Two distinct PFGE patterns were observed among 44S. Enteritidis isolates. There was no association between virulence profiles or antibiotypes and PFGE clusters. CONCLUSIONS: Overall, this study provides valuable information on the virulence gene content, antibiotic resistance and genetic diversity of S. Enteritidis isolated from human sources in Iran.