The role of Staphylococcus aureus FadB in resistance to bile

Resistance to the bactericidal effects of bile is crucial for the survival of Staphylococcus aureus in the human gut. This study was conducted to identify and characterize components of the bacteria, which allow it to resist bile acids. A comparative study was used to investigate the natural protein diversity within the Staphylococcus in relation to bile resistance. Imaging of one-dimension gel electrophoresis showed a unique protein band in samples prepared from bile-treated S. aureus. Mass spectrometry and database analysis showed the protein to be FadB. which has a role in lipid metabolism in Escherichia coli. It is hypothesized that fadB was responsible for the observed bile salt resistance phenotype; to test this, a ΔfadB strain was created in S. aureus SH1000. The mutant phenotype showed a significant decrease in viability upon exposure to bile acids in comparison with the parental wild type. Furthermore, survival of S. aureus ΔfadB was attenuated in an in vitro human colonic model, implicating fadB in S. aureus colonization of the human intestine. Moreover, upregulated expression of fadB was detected upon exposure to bile salts. To further confirm the role of FadB in bile salt resistance, the gene was cloned under the control of an inducible promoter, which enabled arabinose-dose dependent expression of fadB in E. coli JW113 as a heterologous host, confirming a bile resistant phenotype. Recombinant FadB was purified and shown to have affinity for cholic acid and might possess an ability to modify bile salts

Using of Integrons as Biomarker to Assess Dissemination and Diversity of Antimicrobial Resistance Genes in Farm Animal Manure

Antimicrobial agents are widely used for treatment of animal and human diseases. Heavy use of antimicrobial agents permits bacteria to develop resistance to these agents specifically when a dose of antibiotic is insufficient or course of treatment is incomplete. Antimicrobial resistance genes (ARGs) are usually associated with mobile genetic elements (MGEs) including Integron therefore; these genes can transmit among bacteria via horizontal transmission. The current study was conducted to assess the possible role of manure in dissemination of antimicrobial resistance. The presence, quantitate, and diversity of resistance genes associated with Integron class 1 have been assessed using conventional and quantitative polymerase chain reaction (PCR) combined with sequencing of gene cassette within Integron and analysis of sequenced data by blast tool. Thirty-eight samples were found a positive for Integron and concentration of Integron in positive sample ranged from from 106-1010 copies/g of manure. High frequencies were detected to genes that encoded to sulphonamide and ammonium compound resistance. These genes were detected in 25% and 23% respectively of the total manure samples. In general, the detected genes in manure functionally belong to five protein families including Efflux pump, DNA repair, heavy metal resistance, membrane protein, and antibiotic resistance. Manure might act as a hotspot from which ARGs emerge and transfer to the environment and then to the animal and human environments

Novel CRISPR/Cas13- based assay for diagnosis of avian infectious bronchitis

Infectious bronchitis is an acute respiratory disease of poultry associated with reduced egg production and heavy economic losses in chicken flocks. Rabid and accurate detection of IB virus (IBV) is essential for controlling and preventing the infection. In this study, we developed a rapid, accurate, and instrument less assay to detect IBV. For the first time, reverse transcription- Recombinase polymerase amplification (RT-RPA) coupled with CRISPR/Cas13 (SHERLOCK) was used to rapidly visualize IBV. The novel assay was tested in timing, sensitivity, and specificity. The spike gene (S gene) was used as a target gene for detecting the virus. Three samples were used to optimize the assay; sample form confirmed infected chickens with IB, positive sample (full synthesis of S gene), and negative sample from free IB infected chickens. The results show that the Sherlock-based Cas13 platform is a highly specificity and sensitivity assay for detecting infectious bronchitis virus. The assay detected ten copies per µL of the input RNA. No false positives or cross-reactions were seen when bovine coronavirus (BCV) was used instead of IBV in the tested sample. Readout of the results needs just fifty minutes, including RNA extraction. Furthermore, No instrument was used, and amplification of the virus's nucleic acid was performed at room temperature. Sherlock-based Cas13 should clinically use for rapid diagnosis of infectious bronchitis in chickens. However, further studies and experiments are needed to perform the assay at the sample base without extraction of RNA

Staphylococcus aureus FadB is a dehydrogenase that mediates cholate resistance and survival under human colonic conditions

Staphylococcus aureus is a common colonizer of the human gut and in doing so it must be able to resist the actions of the host’s innate defences. Bile salts are a class of molecules that possess potent antibacterial activity that control growth. Bacteria that colonize and survive in that niche must be able to resist the action of bile salts, but the mechanisms by which S. aureus does so are poorly understood. Here we show that FadB is a bile-induced oxidoreductase which mediates bile salt resistance and when heterologously expressed in Escherichia coli renders them resistant. Deletion of fadB attenuated survival of S. aureus in a model of the human distal colon

Antibiotic resistance genes in farm animal slaughterhouse wastes in Al-Dewanyiah province, Iraq

Environment represents as a reservoir for emerging and disseminating of antibiotic resistance genes. Slaughterhouse waste is one of the important sources of antibiotic resistance genes (ARGs) even after treatment processes. This study was conducted to evaluate role of farm animal slaughterhouse in dissemination of antibiotics resistance in Al-Dewanyiah, Iraq. A total of eighty samples were collected from the central farm animal slaughterhouse. The detection was based on three mobile genetic elements and nine antibiotic resistance genes. The results showed that tetO & tetK are common resistance genes in the tested samples with great relative abundance 60%. While, MGE transposon (Tn3) was detected in 80% of the tested samples. Gene encoding resistance to quinolone, methicillin, aminoglycoside and β-lactamases were also detected in the tested samples. Presence of three class of integrons as a mobile genetic were tested and the results of type 1 recorded high abundance (P>0.05) as a compare with type 2 and type 3 integrons. Furthermore, concentrations of ARGs and MGEs per gram of sample were tested using qPCR. The genes encoding for tetracycline resistance and transposon (Tn3) were found in higher concentration (P>0.05) (copy number) per gram of slaughterhouse sediments comparing with the selected genes. Quantification of ARGs and MGEs in the slaughterhouse wastes indicates that those wastes represent as a hotspot for dissemination of antibiotic resistance to the environment. Low darning and no treatment for slaughterhouse wastes might increase abundance of ARGs and resistant bacteria in the natural environment

Evaluation of efflux pump inhibitory activity of some plant extracts and using them as adjuvants to potentiate the inhibitory activity of some antibiotics against Staphylococcus aureus

Background: Antibiotic resistant pathogens became a real global threat for human and animal health. This needs to concentrate the efforts to minimise and control these organisms. Efflux pumps are considered as one of the important strategies used by bacteria to exclude the harm materials out of the cell. Inhibition of these pumps can be an active strategy against multidrug resistance (MDR) pathogens. There are two source of efflux pump inhibitors can be used, chemical and natural inhibitors. The chemical origin efflux pump inhibitors have many toxic side effects while the natural origin characterized by a wide margin of safety for the host cell. Aim: In this study the ability of some plant extracts like (propolis show rosemary, clove, capsaicin and cumin) to potentiate the inhibitory activity of some antibiotics as (ciprofloxacin, erythromycin, gentamycin, tetracycline and ampicillin) against S. aureus pathogen were tested. Methods: Efflux pump inhibitory activity of the selected plant extracts were tested using ethidium bromide accumulation assay. Results: The results have shown that Propolis has a significant synergistic effect in combination with ciprofloxacin, erythromycin and gentamycin. While, it has no effect with tetracycline or ampicillin. Also, no synergic effect was noticed in combination of the MIC for the selected plant extracts (rosemary, clove, capsaicin and cumin) with any of the tested antibiotics. Interestingly, according to the results of EtBr accumulation assay, Propolis has potent inhibitory activity against S. aureus (MRS usa300) pump system. Conclusion: This study suggests that Propolis might act as a resistance breaker that is able to restore the activity of ciprofloxacin, erythromycin and gentamycin against S. aureus strains, in case of the Efflux-mediated antimicrobial resistance mechanisms