Genetics

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Autolytic characterization of selected Enterococcal strains, (previously Streptococcal)
(2007) Sukkhu, Melisha.; Beukes, Mervyn.
Autolysins are enzymes that cleave specific structural components within the bacterial cell wall. They contribute to numerous cellular activities such as cell growth, cell division, peptidoglycan recycling and turnover. In this study, twelve Enterococcal isolates (previously from the genus Streptococcus) were examined for susceptibility to the antibiotics Penicillin G and Vancomycin, using a Disk Diffusion and a Microtitre plate assay. In both methods, all twelve strains were resistant to Vancomycin. Six of these strains were susceptible to Penicillin G. The minimum inhibitory concentration (MIC) values were twice that of the disk diffusion assay values. In the presence of antibiotic, the growth rates for the six strains were halved. Autolysins were extracted from the respective cell cultures using a 4% SDS precipitation method. The protein concentrations were calculated and estimated to be within the range of 5.47- to 6.35 μg/μl. Profiles of the SDS precipitate were analyzed on SDS-PAGE. Autolytic proteins were identified and partially analyzed by renaturing SDS-PAGE (zymograms) using gels containing cell wall substrate. Seven lytic bands of molecular weights 25, 30, 50, 63, 75 95 and 145 kDa (designated Autolysin A to G, respectively) were selected for further analysis. The temporal distribution of the enzymes ranged from the mid exponential phase to the early death phase. The seven proteins were blotted onto polyvinylidene difluoride (PVDF) membranes and excised for N-terminal sequencing. Blast analysis of the respective N-terminal sequences showed autolysins A, C, D, E and F to have 100% similarity to the muramidase, amidase and peptidase from S. cremoris, S. suis, S. pneumonia, S. pyogenes and E. faecium, respectively. Biochemical characterization confirmed autolysins A, B, E and F to exhibit muramidase activity, and autolysin C and G to exhibit peptidase activity. Autolysin D displayed 100% similarity to the protein LytA, a peptidoglycan hydrolase that is known to exhibit amidase activity. Blast analysis could not determine any significant similarities for autolysins B and G to previously identified autolysins, thus indicating they may perhaps be novel autolysins.
Characterisation of antibiotic resistance in Streptococcus, Enterococcus and Staphylococcus using a bioinformatics approach.
(2005) Ramsuran, Veron.; Beukes, Mervyn.
The rate at which bacterial pathogens are becoming resistant to antibiotics is quite alarming, and therefore much attention has been focussed on this area. The mechanism whereby the bacterial cells acquire resistance is studied in order to determine how this process works as well as to determine if any future resistance mechanisms can be circumvented. In this study three different genera and the antibiotics that are resistant to them were used, namely, penicillin resistant Streptococcus, vancomycin resistant Enterococcus and methicillin resistant Staphylococcus. The results prove that the active sites SXXK, SXN and KT(S) G in the penicillin resistance Streptococcus plays a major role in resistance. It is seen in this study that the SXXK active site is found in all the resistant and most of the intermediate strains, therefore proving to be an important component of the cell wall resistance. It was subsequently noticed the greater the number of mutations found in the sequences the higher the resistance. Three dimensional structures showed the actives sites and their binding pockets. The results also show the change in conformation with a mutation in the active site. The results also proved that the Penicillin Binding Protein (PBP) genes essential for resistance are PBP Ia, PBP 2b and PBP 2x. The results obtained, for the vancomycin resistance in Enterococcus study, proved that the VanC and VanE cluster are very much alike and VanE could have evolved from VanC. There is also close similarity between the different ligase genes. The VanX 3D structure shows the position of the critical amino acids responsible for the breakdown of the D-Ala-D-Ala precursors, and the VanA ligase 3D structure shows the amino acids responsible the ligation of the D-Ala-D-Lac precursors. The analysis performed on the methicillin resistance in Staphylococcus study showed that the genes used to confer resistance are very similar between different strains as well as different species.
Genetic characterization of resistance and virulence genes in Enterococcus species from animal isolates in Durban.
(2018) Eberechi, Phoebe Nnah.; Adeleke, Matthew Adekunle.; Zishiri, Oliver Tendayi.
Misuse of antimicrobials in animal agriculture has given rise to strains of bacteria that are resistant to multiple antibiotics. Enterococci bacteria have emerged among such antibiotic-resistant strains of bacteria and infections due to antibiotic-resistant bacteria is one of the world’s critical health challenge. Enterococci are gut commensal bacteria but are currently confirmed pathogenic bacteria responsible for so many hospital-acquired infections like urinary tract infections. The aim of this research was to detect the occurrence of Enterococcus species in chickens, cats, and dogs; their phenotypic and genotypic resistance to antibiotic drugs and virulence genes. Isolation of Enterococcus species was done using microbiological culture methods and confirmed using specific primers through Polymerase Chain Reaction (PCR). Presumptive Enterococcus growth on bile esculin agar was positive for 94% of all the isolates. Overall, 77.3% of the isolates were positive for Tuf gene (Enterococcus genus-specific gene). Enterococcus faecalis was detected at a higher frequency (40.4%; P <0.05) compared to Enterococcus faecium (8.5%). All the Enterococcus isolates were susceptible to High-Level Gentamicin on antimicrobial susceptibility test. Enterococcus species in chickens exhibited higher resistance to the antibiotics than the pets. Highest resistance was observed in Quinupristin/Dalfopristin (89.4%) followed by Vancomycin (87.9%), Rifampicin (85%), Ampicillin (76.6%), Erythromycin (72.3%), and Tetracycline (64.5%). Chloramphenicol (24.8%), High-Level Streptomycin Resistance (24.1%), and Ciprofloxacin (14.2%). Eighty-four percent (84%) of the Enterococcus isolates expressed multidrug resistance (MDR). Three of the four resistance genes screened were detected: 21.3%, 7.8% and 4.3% for Kanamycin, Streptomycin, and Vancomycin resistance genes respectively. Gentamicin resistance gene was absent in all the isolates. PCR detection of virulence gene showed highest prevalence in EfaA gene at 88.7% frequency followed by GelE (82.3%), ccf (81.6%), Esp (26.2%) and CylA (25.5%). All E. faecalis and E. faecium detected harbored multiple virulence genes. These findings show that chickens, cats, and dogs can be colonized by pathogenic Enterococci which harbor resistance and virulence genes and are multidrug resistant. It is therefore important that antibiotics are used prudently in animal husbandry to mitigate emergence and transfer of Enterococci pathogens to humans via food chain and direct contact of pets by their owners.

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Browsing Genetics by Subject "Antibiotics."