Bacteriological and Molecular Studies on the Enterococcus Species Isolated From Diseased Fish and Its Effect on Fish Farm Profits

The study was carried out on 120 fish samples O. nitoticus collected from Kafir El-Sheikh Governorate (60 diseased and 60 apparently healthy fish). The clinical picture of naturally infected O. niloticus showed haemorrhagic spots on the operculum, base of fins and mouth edges, skin darkening, uni-or bJJateraJ exophthalmia and skeletal deformity in some cases abdominal distension was observed. Postmortum lesions in O. niloticus revealed congested and enlarged liver or pale with grayish nodules in some cases. Spleen and kidneys were enlarged and congested and abdominal cavity contained serous fluid in some cases. Bacteriological examination revealed the isolation of (26) streptococcus isolates with an incidence of (43.3%) from diseased O. niloticus, and isolation of (17) isolates with an incidence of (28.3%) from the 60 apparently healthy . These isolates were biochemically tested. SDS-P AGE analysis of whole cell protein of selected serotyped strains revealed the presence of 7-13 protein bands and the most common characteristic bands were 36.67 KDa, 27.37 KDa and 44.0 KDa. Kb. DNA profile analysis of the 3 streptococcus species showed common band at 321 Serological examination of 37 selected isolates result in differentiation into 17 Enterococcus faecalis, 12 Streptococcus iniae, 5 Streptococcus pneumoniae and 3 untypeable strains. Experimental infection of 8 groups of O. niloticus (each of 10 fish) with bacterial suspension of 8 isolates (2 Enterococcus faecalis, 5 Streptococcus iniae and 1 Streptococcus pneumoniae result in mortality rate of 20%, 10% and 0%, respectively. While, inoculation of the bacterial filtrate of the same isolates result in mortality rate of 30%, 22% and 10%. Our results cleared that the enterococci causes a great economic losses to fish farm production and it differ according to the type of bacteria that infected the fish. In bacterial suspension infection the weight losses for each 100/fish were 450 gm, 262.5 gm and zero losses zero losses for S. fecalis, S. iniae and S. pnumoniae and the return losses reached to 4.5 LE, 2.62 LE and zero losses for S. fecalis, S. iniae and S. pnumoniae. While, in bacterial filtrate the weight losses for each 100/fish were 675 gm, 1237.5 and 112.5 gm for S. fecalis, S. iniae and S. pnumoniae and the return losses reached to 6.75 LE, 12.37 LE and 11.25 losses for S. fecalis, S. iniae and S. pnumoniae. From these results we concluded that: Fish farms should avoid use of polluted water. Fish handlers with cut wounds should avoid fish handling without gloves as Streptococcus soft tissue causing sepsis, infection endocardities, urinary tract infections, labor pneumonia and meningitis. Human should keep water sources away from sewage pollution

Bacteriological and Molecular Studies on the Enterococcus Species Isolated From Diseased Fish and Its Effect on Fish Farm Profits

The study was carried out on 120 fish samples O. nitoticus collected from Kafir El-Sheikh Governorate (60 diseased and 60 apparently healthy fish). The clinical picture of naturally infected O. niloticus showed haemorrhagic spots on the operculum, base of fins and mouth edges, skin darkening, uni-or bJJateraJ exophthalmia and skeletal deformity in some cases abdominal distension was observed. Postmortum lesions in O. niloticus revealed congested and enlarged liver or pale with grayish nodules in some cases. Spleen and kidneys were enlarged and congested and abdominal cavity contained serous fluid in some cases. Bacteriological examination revealed the isolation of (26) streptococcus isolates with an incidence of (43.3%) from diseased O. niloticus, and isolation of (17) isolates with an incidence of (28.3%) from the 60 apparently healthy . These isolates were biochemically tested. SDS-P AGE analysis of whole cell protein of selected serotyped strains revealed the presence of 7-13 protein bands and the most common characteristic bands were 36.67 KDa, 27.37 KDa and 44.0 KDa. Kb. DNA profile analysis of the 3 streptococcus species showed common band at 321 Serological examination of 37 selected isolates result in differentiation into 17 Enterococcus faecalis, 12 Streptococcus iniae, 5 Streptococcus pneumoniae and 3 untypeable strains. Experimental infection of 8 groups of O. niloticus (each of 10 fish) with bacterial suspension of 8 isolates (2 Enterococcus faecalis, 5 Streptococcus iniae and 1 Streptococcus pneumoniae result in mortality rate of 20%, 10% and 0%, respectively. While, inoculation of the bacterial filtrate of the same isolates result in mortality rate of 30%, 22% and 10%. Our results cleared that the enterococci causes a great economic losses to fish farm production and it differ according to the type of bacteria that infected the fish. In bacterial suspension infection the weight losses for each 100/fish were 450 gm, 262.5 gm and zero losses zero losses for S. fecalis, S. iniae and S. pnumoniae and the return losses reached to 4.5 LE, 2.62 LE and zero losses for S. fecalis, S. iniae and S. pnumoniae. While, in bacterial filtrate the weight losses for each 100/fish were 675 gm, 1237.5 and 112.5 gm for S. fecalis, S. iniae and S. pnumoniae and the return losses reached to 6.75 LE, 12.37 LE and 11.25 losses for S. fecalis, S. iniae and S. pnumoniae. From these results we concluded that: Fish farms should avoid use of polluted water. Fish handlers with cut wounds should avoid fish handling without gloves as Streptococcus soft tissue causing sepsis, infection endocardities, urinary tract infections, labor pneumonia and meningitis. Human should keep water sources away from sewage pollution

A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria

Efforts to develop live attenuated vaccines against Mycobacterium avium subspecies paratuberculosis (Map), using indirect methods to screen Map deletion mutants for potential efficacy, have not been successful. A reduction in the capacity to survive in macrophages has not predicted the ability of mutants to survive in vivo. Previous studies for screening of three deletion mutants in cattle and goats revealed one mutant, with a deletion in relA (ΔMap/relA), could not establish a persistent infection. Further studies, using antigen presenting cells (APC), blood dendritic cells and monocyte derived DC, pulsed with ΔMap/relA or a 35 kDa Map membrane protein (MMP) revealed a component of the response to ΔMap/relA was directed towards MMP. As reported herein, we developed a bacterium viability assay and cell culture assays for analysis and evaluation of cytotoxic T cells generated against ΔMap/relA or MMP. Analysis of the effector activity of responding cells revealed the reason ΔMap/relA could not establish a persistent infection was that vaccination elicited development of cytotoxic CD8 T cells (CTL) with the capacity to kill intracellular bacteria. We demonstrated the same CTL response could be elicited with two rounds of antigenic stimulation of APC pulsed with ΔMap/relA or MMP ex vivo. Cytotoxicity was mediated through the perforin granzyme B pathway. Finally, cognate recognition of peptides presented in context of MHC I and II molecules to CD4 and CD8 T cells is required for development of CTL

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

High Prevalence of ESBL and Plasmid-Mediated Quinolone Resistance Genes in Salmonella enterica Isolated from Retail Meats and Slaughterhouses in Egypt

The emergence and spread of multidrug-resistant Salmonella enterica (S. enterica) to humans through food of animal origin are considered a major global public health concern. Currently, little is known about the prevalence of important antimicrobial resistance genes in S. enterica from retail food in Africa. Therefore, the screening and characterization of the extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes in S. enterica isolated from retail meats and slaughterhouses in Egypt were done by using PCR and DNA sequencing techniques. Twenty-eight out of thirty-four (82.4%) non-duplicate S. enterica isolates showed multidrug-resistance phenotypes to at least three classes of antimicrobials, and fourteen (41.2%) exhibited an ESBL-resistance phenotype and harbored at least one ESBL-encoding gene. The identified β-lactamase-encoding genes included blaCTX-M-1, blaCTX-M-3, blaCTX-M-13, blaCTX-M-14, blaCTX-M-15, and blaSHV-12 (ESBL types); blaCMY-2 (AmpC type); and blaTEM-1 and blaOXA-1 (narrow-spectrum types). PMQR genes (included qnrA, qnrB, qnrS, and aac(6′)-Ib-cr) were identified in 23 (67.6%) isolates. The presence of ESBL- and PMQR-producing S. enterica with a high prevalence rate in retail meats and slaughterhouses is considered a major threat to public health as these strains with resistance genes could be transmitted to humans through the food chain

Risk of Staphylococcus aureus Isolated from Poultry Meat of Chicken with Arthritis in Poultry Farms

Staphylococcus aureus is a major pathogen that affects both people and animals. Staphylococcus aureus causes food poisoning in addition to invasive diseases as arthritis and septicemia. This study was done on 70 chicken samples obtained from 7 different farms of chickens with symptoms of arthritis in Kafr El-sheikh government, Egypt. In this study out of 70 samples of chickens from different farms, 37 (52.8%) samples were recognized as coagulase-positive staphylococci (CoPS) and 33 (47.1%) were recognized as coagulase-negative staphylococci (CoNS). By using the microtitre plate method, seven out of 37 (18.9%) CoPS were positive for biofilm production with variable degrees. The pattern of antibacterial sensitivity of 7 Staphylococcus aureus isolates against 12 commercially available antibiotic discs showed 100 % resistance to oxytetracycline then Amoxicillin (71.43%), Erythromycin (57.14%), Norfloxacin (14.29%), Tetracycline (42.86), Sulphamethoxazole (42.86%), Gentamicin (42.86%), Ampicillin (42.86%), kanamycin (28.57), cephatotin (28.57), doxycycline (0%) and the least was observed with chloramphenicol (0%). seven of positive S. aureus isolates were introduced in order to identify the staphylococcal enterotoxin genes, SEA, SEB, SEC, SED, and SEE and integron by PCR test Which 4 out of 7 isolates (57.1 %) were positive for SEB and SED only while were other isolate were negative for all SE gene. Class 1 integron cassettes were detected in 6 isolates from 7 (85.7%) of tested isolates. In conclusion, this is the first study to report the detection and identification of enterotoxin and class 1 integron in S. aureus isolated from poultry meat of chicken that suffered from arthritis.

Effect of Silver Nanoparticles on Biofilm Formation by Clostridium perfringens Isolated from Poultry and Molecular Typing of Strains by ERIC-PCR

The aim of this is study was to evaluate of antibacterial activity of silver nanoparticles (AgNPs) on multidrug resistant (MDR) Cl. perfringens strains isolated from broilers as an alternative to conventional antibiotics and Molecular typing of the C. perfringens strains using ERIC-PCR for assessment of genetic relationship between strains isolated from different organs. A total of 20 isolates of Cl. perfringens were isolated from necrotic enteritis affected poultry located in El-Behera Governorate, Egypt. Antimicrobial resistance pattern was evaluated. ERIC- PCR genotyping of Cl. perfringens   isolates was performed to detect the genetic diversity and the degree of similarity between isolates. Antibacterial activity of AgNPs on MDR strains was evaluated by the minimum inhibitory concentration (MIC) and, minimum bactericidal concentration (MBC). Transmission Electron Microscopy (TEM) imaging was conducted to evaluate the effect of silver nanoparticles on C. perfringens cells. Scanning Electron Microscopy (SEM) imaging used to evaluate the effect of AgNPs on Cl. perfringens biofilm formed on glass cover slide for different periods of incubation with the same concentration of silver nanoparticles. Out of 85 collected samples from broiler chicken farms, 20 isolates of Cl. perfringens type A were isolated from broiler chickens at El-Behera governorate, Egypt. Most of isolates were MDR. ERIC PCR genotyping classified the isolates into 4 clusters (C1-C4).  MBC was 30ug/ml, while MIC was 20ug/ml and the bacterial growth completely inhibited after 24 hours. TEM and SEM images showed that AgNPs has exerted broad-spectrum bactericidal activity against MDR Cl. perfringens isolate and against biofilm.  

Genetic Relationship between Salmonella Isolates Recovered from Calves and Broilers Chickens in Kafr El-Sheikh City Using ERIC PCR

A prevalent bacterial intestinal infection with severe economic damage is salmonellosis. Our study was carried out to diagnose Salmonella from chickens and calves, to determine its resistance to antimicrobials’ phenotypic and genotypic characterization of integrons and β lactamase genes in the multidrug resistance of different Salmonella serotypes, and to detect the genetic relationship between Salmonella isolates collected from different origins using an ERIC PCR. In total, 200 samples from diseased chicken and diarrheic calves were obtained from 50 various farms from Kafr El-sheikh, Egypt. Salmonella poultry isolates were characterized as S. Typhimurium (3/8), S. Enteritidis (3/8), and S. Kentucky (2/8), but Salmonella isolates from cattle were S. Enteritidis (1/2) and S. Kentucky (1/2). When antibiotic susceptibility testing was completed on all of the isolates, it showed that there was multidrug resistance present (MDR). A PCR was applied for identifying the accompanying class 1 integrons and ESBLs from MDR Salmonella isolates (two isolates of S. Kentucky were divided as one from calf and one from poultry). Our results detected blaTEM and class 1 integron, but were negative for bla IMP, bla VIM, and bla SHV. An ERIC PCR was conducted for understanding the clonal relation between various β-lactamase-producing MDR Salmonella isolates. The same four previously mentioned isolates were also tested. The two isolates of S. Enteritidis isolated from poultry and calves had 100% similarity despite indicating that there were interactions between broilers and calves living on the same farm that caused infection from the same Salmonella strains, while the other two isolates of S. Kentucky showed only 33% serovarities

A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria

Efforts to develop live attenuated vaccines against Mycobacterium avium subspecies paratuberculosis (Map), using indirect methods to screen Map deletion mutants for potential efficacy, have not been successful. A reduction in the capacity to survive in macrophages has not predicted the ability of mutants to survive in vivo. Previous studies for screening of three deletion mutants in cattle and goats revealed one mutant, with a deletion in relA (ΔMap/relA), could not establish a persistent infection. Further studies, using antigen presenting cells (APC), blood dendritic cells and monocyte derived DC, pulsed with ΔMap/relA or a 35 kDa Map membrane protein (MMP) revealed a component of the response to ΔMap/relA was directed towards MMP. As reported herein, we developed a bacterium viability assay and cell culture assays for analysis and evaluation of cytotoxic T cells generated against ΔMap/relA or MMP. Analysis of the effector activity of responding cells revealed the reason ΔMap/relA could not establish a persistent infection was that vaccination elicited development of cytotoxic CD8 T cells (CTL) with the capacity to kill intracellular bacteria. We demonstrated the same CTL response could be elicited with two rounds of antigenic stimulation of APC pulsed with ΔMap/relA or MMP ex vivo. Cytotoxicity was mediated through the perforin granzyme B pathway. Finally, cognate recognition of peptides presented in context of MHC I and II molecules to CD4 and CD8 T cells is required for development of CTL

Isolation and Molecular Characterization of <i>Corynebacterium pseudotuberculosis</i>: Association with Proinflammatory Cytokines in Caseous Lymphadenitis Pyogranulomas

Corynebacterium pseudotuberculosis (C. pseudotuberculosis) is a causative agent of numerous chronic diseases, including caseous lymphadenitis (CLA) in sheep and goats, which has a zoonotic potential in humans in addition to a poor therapeutic response. In this study, out of 120 collected samples, only 12 (10%) were positive for C. pseudotuberculosis by PCR and by intraperitoneal injection of male Guinea pigs and then characterized for antimicrobial susceptibility and its genetic-relatedness by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), which showed 2–4 bands ranging from 100 to 3000 bp that can be clustered into four clusters (C1–C4). Despite the serotype biovar 1 only infecting sheep and goats, ERIC–PCR reveals intra-subtyping variation. Examination of affected LNs and organs revealed marked enlargement with either thick creamy green pus or multiple abscesses of variable sizes with a central caseated core surrounded by dense fibrous capsule. A histopathological examination revealed a central necrotic core surrounded by a peripheral mantle of mononuclear cells and a fibrous capsule. Positive immune expression of nuclear factor kappa B (NF-κB/p65) and interleukin-1β (IL-1β) and negative expression of tumor necrosis factor (TNF) in CLA is the first report to our knowledge. Conclusion: In CLA pyogranulomas, IL1β is a more crucial proinflammatory cytokine than TNF in the regulation of C. pseudotuberculosis infection, which is accompanied by marked NF-κB immunoexpression. Therefore, the NF-κB/p65 signaling pathway is involved in the activation of IL1β, and additional immunohistochemical studies are required to determine the various roles of NF-κB/p65 in the inflammatory response within CLA pyogranulomas to control this pathogen