Molecular characterization of Aeromonas hydrophila isolates from diseased fishes in district Kasur, Punjab, Pakistan

Pakistan is an agricultural country and fisheries play a very important role in the economic development of the country. Different diseases are prevalent in Pakistani fish but information related to the causative agents is not well-known. Keeping in view the significance of bacterial pathogens as the causative agents of multiple fish diseases, the present study was conducted for identification, characterization and analysis of virulence genes of Aeromonas spp. isolated from diseased fishes. A total of fifty fish samples having multiple clinical indications were collected from different fish farms of district Kasur, Punjab Pakistan. For isolation of Aeromonas spp. samples were enriched and inoculated on Aeromonas isolation medium. Isolates were identified and characterized by different biochemical tests, Analytical Profile Index (API) 20E kit and Polymerase Chain Reaction (PCR) assays. All isolates were screened for three putative virulence genes including aerolysin (aer), haemolysin (hyl) and heat labile cytotonic enterotoxin (alt). Seven isolates of Aeromonas (A.) hydrophila were retrieved and identified based on API 20E. These isolates were further confirmed as A. hydrophila on the basis of PCR assays. Three isolates were detected positive for the presence of virulence genes (alt and hyl). Whereas aerolysin (aer) gene was not present in any of A. hydrophila isolates. The present study confirmed A. hydrophila as the causative agent of epizootic ulcerative syndrome and motile Aeromonas septicemia in fish farms of district Kasur, Punjab Pakistan. Moreover, detection of two virulence genes (alt and hyl) in A. hydrophila isolates is a threat for fish consumers of study area.

Antibacterial activity of Ricinus communis plant extract against antibiotic resistant Helicobacter pylori and Gluconobacter oxydans isolated from fresh apple juices samples

Bacteria were isolated from samples of Fresh Apple juices from shops of three different localities of Lahore. Analysis of samples from Liberty, Anarkali and Yateem khana Markets show different levels of contamination. There were pathogenic and non-pathogenic bacteria in all samples and were identified by the morphological and biochemical tests. Most of the plasmids of pathogenic bacteria were 4kb in their molecular size. Ribotyping of 16S ribosomal RNA gene sequencing was done to confirm Helicobacter pylori strain and Gluconobacter oxydans. The highest sensitivity of 210mm was shown by Enterobacter sp. against Aztheromysine disk (15µg) while Micrococcus sp. was highly resistant against all of the Antibiotics applied. The antibiotic resistance of pathogenic bacteria was also checked against Ricinus communis plant's extracts, all isolated bacterial pathogens were resistant but only, E.coli was inhibited at 300µl of the extracts. Presence of pathogenic bacteria in Apple juice samples was due to contamination of sewage water in drinking water while some of these pathogenic bacteria came from Apple's tree and other from store houses of fruits.

Potential of Sambiloto (Andrographis paniculata Nees) Extract against Salmonella Pullorum

Background: Salmonella Pullorum are pathogenic bacteria that causes salmonellosis and causes heavy economic losses in the poultry industry and are zoonotic. Treatment of diseases caused by bacteria generally use antibiotics, but excessive administration of antibiotics causes bacterial resistance and residues in livestock. Major chemical constituents of Sambiloto are andrographolide and flavonoids. Andrographolide has antibacterial effect in addition to being antitoxic, anticancer, anti-inflammatory and antiallergic. Methods: The research was conducted by isolating and identifying Salmonella Pullorum on SSA media and a series of biochemical tests (TSIA, SIM, SCA, urea media and sugar test), manufacturing sambiloto extract, testing the sensitivity of several antibiotics using the disk diffusion method and testing the activation of sambiloto extract against Salmonella Pullorum using the disk diffusion and dilution methods. Result: The result show that sambiloto had antibacterial activity because it contained andrographolide, flavonoids, saponins, alkaloids and tannins and the lowest extract dose that effectively killed Salmonella Pullorum is concentrations of 20%.

Isolation, antimicrobial susceptibility patterns, and risk factors assessment of non-typhoidal Salmonella from apparently healthy and diarrheic dogs

Background Dogs are one of the important asymptomatic carriers of antimicrobial resistant and potentially pathogenic strains of Salmonella. They can harbor large bacterial load in the intestines and mesenteric lymph nodes which can be shed in their feces with the possibility of transmission to humans. Therefore, a cross-sectional study was conducted with the objectives of estimating the prevalence of non-typhoidal Salmonella, assessing the risk factors for dog’s Salmonella carriage, and profiling the antimicrobial resistance pattern of Salmonella isolates among housed dogs in Harar town, Eastern Ethiopia. A total of 415 rectal swab samples were collected from randomly selected dogs. Samples were examined for non-typhoidal Salmonella using standard bacteriologic culture and biochemical tests. The disk diffusion method (Kirby-Bauer test) was employed to evaluate the isolates for their susceptibility against five antimicrobials. Results Non-typhoidal Salmonella were isolated from 26 (6.3%) of the rectal swab samples, with significantly higher occurrence in diarrheic (15.2%) than non-diarrheic (5.5%) dogs. The risk of Salmonella harboring was significantly higher in female dogs than in male dogs (OR = 2.5, p = 0.027). Dogs fecal shedding of Salmonella was relatively higher in households who used offal as a main feed type for their dogs (23.1%; 95% CI = 5–53.8) than those who used leftover food (10.1%; 95% CI = 5.7–16.1) and practiced mixed feeding system (17%; 95% CI = 7.6–30.8). Salmonella isolates showed higher resistance to ampicillin (41.7%), while all isolates were fully susceptible to gentamicin. Moreover, 58.3% of Salmonella isolates showed resistance to at least one of the tested antimicrobials. Majorities (72.7%) of the dog owners had no awareness on the risk of zoonotic salmonellosis from dog and all of the respondents use bare hand to clean dog kennel. Conclusion Our study reveals the importance of both diarrheic and apparently healthy housed dogs in the harboring and shedding of antimicrobial resistant non-typhoidal Salmonella. The risk of non-typhoidal Salmonella spread among pet owners is not negligible, especially in households who use offal as main feed type. Therefore, an integrated approach such as: proper dog handling practices; continuous evaluation of antimicrobial resistance; and rational use of antimicrobials in the field of veterinary sector are necessary to tackle the problem.

Hematological and Serum Biochemical Changes and Their Prognostic Value in Horses Spontaneously Poisoned by Crotalaria spectabilis

Determining the prognosis of poisoning by plants containing pyrrolizidine alkaloids is usually challenging. This study aimed to identify important prognostic parameters that can determine the severity of spontaneous poisoning by Crotalaria spectabilis in horses. Blood samples from 42 horses spontaneously poisoned by oats contaminated with C. spectabilis seeds were evaluated. Complete blood counts (CBC) and serum biochemical tests [urea, creatinine, total protein, albumin, total and direct bilirubin concentrations, aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), and creatine kinase (CK) activities] were performed. Horses were followed up for 12 months to determine the long-term survival rate; after 12 months, they were divided into two groups: survivors (n = 30) and non-survivors (n = 12). Horses spontaneously poisoned with C. spectabilis had higher levels of urea, globulin, bilirubin (total, direct, and indirect), AST, GGT, and CK than the reference values. Non-survivor horses showed significantly higher (p < 0.05) values of hemoglobin, GGT, and direct bilirubin than the survivor horses. Horses with serum GGT activity higher than 95 U/l had 14.0 times the risk of death compared to animals showing activities equal to or lower than this value, whereas horses with serum direct bilirubin concentration higher than 0.6 mg/dl (10.26 μmol/L) had 5.78 times the risk of death compared to the others. In summary, serum GGT activity and direct bilirubin concentration may be useful prognostic indicators for assessing the severity of C. spectabilis-poisoned horses.

Phenotypic profile of priority multiresistant Аcinetobacter baumannii sequence types (ST 1167, ST 944, ST 208)

Introduction. About 1,000,000 cases of infections caused by Acinetobacter spp. per year are registered globally, making up 1.8% of all the cases of hospital-acquired infections. In compliance with long-term studies carried out in in this country and abroad, Acinetobacter baumannii is a clinically important representative of the Acinetobacter genus. Intraspecific typing of microorganisms is an integral part of a clinical microbiologist's contribution to scoring the outbreaks of purulent-septic infections within the sphere of HAI surveillance. Most of the practicing microbiological laboratories cannot use genotypic typing methods because of their high costs.Objective. Developing a test panel for intraspecific identification of A. baumannii sequence types (ST 1167, ST 944, ST 208) based on their phenotypic properties.Materials and methods. Intraspecific membership of 74 A. baumannii strains obtained from four multipurpose health settings of a large industrial centre was studied using a genetic method (multilocus sequence typing) and a suite of phenotypic methods (biochemical tests, biofilmogenous capacity, growth inhibition zones to antibacterial drugs, sensitivity to aniline dyes, disinfectants and Acinetobacter bacteriophage) was studied.Results. Phenotypic features of three predominant A. baumannii sequence types (ST 1167, 944, 208) were determined.Discussion. An efficacious economy set of differentiating tests allowing identification of intraspecific features of A. baumannii multiresistant strains was сreated.Conclusion. The test panel will enable the laboratories that cannot use sequencing methods to conduct intraspecific differentiation of common A. baumannii sequence types as part of microbiological monitoring.

Molecular characterization of xanthan gum producing Xanthomonas Campestris isolated from dark rot spotted leaves in Keffi, Nasarawa State, Nigeria

Background: Despite the wide application of Xanthan gum, its commercial production remains a global challenge. In recent years, considerable research has been carried out using agro-industrial wastes, which are renewable and abundantly available to produce value-added products. The present study was set out for molecular identification of Xanthomonas campestris from leaves of four different plants with indications of dark rot spots and evaluation of their xanthan gum production capacity. Methods: Twenty-five (25) samples of leaves from four different plants with indications of dark rot spots were collected from the study area and isolated for Xanthomonas campestris following standard microbiological methods. Cultural, morphological and biochemical tests were conducted to confirm the organism. Results: The results revealed that of the total 100 samples taken, 6 leaves (24%) were infected with Xanthomonas species in mint, 3(12%) were infected in mango, 1(4%) were infected in rice and 2(8%) were infected in pepper. Further molecular identification of the isolates was carried out to reveal Xanthomonas campestris pv. vesicatoria strain 85-10 and Xanthomonas perforans strain 91-118. These were further used for the production of xanthan gum using sugar cane molasses substrates extracted from sugar cane, which was used as fermentation medium for the production. Isolates from plants varying ability in Xanthan gum production, with the mint plant having the highest Xanthan gum production (0.10 ± 0.02 to 0.9 ± 0.00 g/l). Conclusion: The present study confirmed the high xanthan gum production capacity of Xanthomonas campestris from dark rot spots containing mint leaves and should be considered during local and industrial production of the xanthan gum

Selenium-Enriched Yeast Relieves Hexavalent Chromium Toxicity by Inhibiting NF-κB Signaling Pathway in Broiler Spleens

This study was conducted to investigate the molecular mechanisms of selenium (Se) antagonism of hexavalent chromium (Cr6+)-induced toxicity. Potassium dichromate (K2Cr2O7) and selenium-enriched yeast (SeY) were used to construct the single Cr6+ and combined Se/Cr6+ exposure broiler models, and then the broilers were randomly divided into four groups (C group, Se group, Se/Cr6+ group, and Cr6+ group). After a 42-day experiment, the spleen tissues of broilers were excised and weighted. The antagonistic mechanisms of Se and Cr6+ were evaluated using histopathological assessment, serum biochemical tests, oxidative stress kits, ELISA, qPCR, and Western blotting. On the whole, there were no significant changes between the C and Se groups. The spleen organ index in the Cr6+ group was significantly decreased, but SeY increased spleen organ index to a certain extent. The levels of SOD and GSH were reduced, and the MDA content was elevated by Cr6+; however, these changes were mitigated by Se/Cr6+ exposure. Importantly, Cr6+ exposure induced a series of histopathological injuries in broiler spleen tissues, while these symptoms were significantly relieved in the Se/Cr6+group. Furthermore, Cr6+ significantly decreased the levels of T-globulin, IgA, IgM, and IgG in serum. Contrarily, dramatically more T-globulin IgA, IgM, and IgG were found in the Se/Cr6+group than in the Cr6+ group. Revealed by the results of qPCR and WB, the expressions of NF-κB, IκBα, and p-IκBα were upregulated in Cr6+ groups, while they were downregulated in Se/Cr6+ group compared to that in Cr6+ group. Besides IFN-γ and IL-2, the expressions of pro-inflammatory cytokines were significantly increased by Cr6+ exposure, but the SeY supplement relived the expression levels mediated by Cr6+ exposure. In conclusion, our findings suggest SeY has biological activity that can protect broiler spleens from immunosuppression and inflammation induced by Cr6+, and we speculate that the NF-κB signaling pathway is one of its mechanisms.

Genome characteristic of Bordetella parapertussis isolated from Iran

Pertussis also known as whooping cough is a respiratory infection in humans particularly in infants and usually caused by Bordetella pertussis. However, Bordetella parapertussis can also cause a similar clinical syndrome. During 2012 to 2015, from nasal swabs sent from different provinces to the pertussis reference laboratory of Pasture Institute of Iran for pertussis confirmation, seven B. parapertussis isolates were identified by bacterial culture, biochemical tests, and the presence of IS1001 insertion in the genome by real-time PCR. Furthermore, the expression of pertactin (Prn) as one the major virulence factor for bacterial adhesion was investigated using western blot. Moreover, the genomic characteristic of one recently collected isolate, IRBP134, from a seven-month infant was investigated using Illumina NextSeq sequencing protocol. The results revealed the genome with G+C content 65% and genome size 4.7 Mbp. A total of 81 single nucleotide polymorphisms (SNPs) and 13 short insertion and deletions were found in the genome compared to the B. parapertussis 12822 as a reference genome showing ongoing evolutionary changes in our isolate. A phylogeny relationship of IRBP134 was also investigated using global B. parapertussis available genomes.

Identification of Leptospira in Patients With Renal Failure Using Serological, Molecular and Pathological Based Techniques, in Shiraz, Iran

Leptospirosis is a relatively rare bacterial infection that affects people and animals caused by pathogenic species of Leptospira. The present study was conducted using nested polymerase chain reaction (PCR), microscopic agglutination test (MAT) and hematological and biochemical tests on 200 blood samples of renal disorders patients in Shiraz, Iran. Also nested-PCR assay and Warthin-Starry (WS) silver staining method was performed on 30 nephrectomised kidney sample. The frequency of pathogenic species of Leptospira infection in patients with renal disorders was 20 % and this infection was significantly correlated with BUN, anemia, RDW, MCV, MCH and hemoglobin levels (P < 0.01). MAT analysis showed that serum samples had positive titers against L. Grippotyphosa (13 samples), L. Ballum (6 sample), L. Pomona (3 samples), L. Canicola (2 samples), L. Icterohaemorrhagiae (1 sample) and L. Hardjo (1 sample) serovars. Twenty-three percent of the kidney samples from the patients with pyelonephritis were infected with the pathogenic species of Leptospira. This study showed that pathogenic Leptospira serovars are present in this area and in patients with renal disorders more attention should be paid to this zoonotic disease.