Pulsed- field gel electrophoresis (PFGE) was first described by Schwartz and Cantor (1) It is now an umbrella term for the alternating of an electric field in more than one direction through a solid matrix to achieve the separation of DNA fragments. The method requires the preparation of unsheared DNA, digestion of the DNA using a rare-cutting restriction endonuclease, separation of fragments by PFGE, and the visualization and interpretation of banding patterns Fig. 1.Conventional agarose gel electrophoresis employs a static field and can resolve DNA fragments up to 50 kilobases (kb), although in practice, fragments larger than 20 kb co-migrate under the conditions usually applied. By introducing a pulse or change in the direction of the electric field, fragments as large as 10 megabases (Mb) can be separated. The time required by DNA fragments of different sizes to reorientate to the new electric field is proportional to their molecular weight and it is this factor that allows the separation and focusing of DNA fragments. Fig 1. Practical steps involved in PFGE.

Pulsed‐Field Gel Electrophoresis

https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(14)61688-8/pdf

Mobile genetic elements and their contribution to the emergence of antimicrobial resistant Enterococcus faecalis and Enterococcus faecium.

Lactobacillus salivarius subsp. salivarius strain UCC118 is a bacteriocin-producing strain with probiotic characteristics. The 2.13-Mb genome was shown by sequencing to comprise a 1.83 Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids. Megaplasmids previously have not been characterized in lactic acid bacteria or intestinal lactobacilli. Annotation of the genome sequence indicated an intermediate level of auxotrophy compared with other sequenced lactobacilli. No single-copy essential genes were located on the megaplasmid. However, contingency amino acid metabolism genes and carbohydrate utilization genes, including two genes for completion of the pentose phosphate pathway, were megaplasmid encoded. The megaplasmid also harbored genes for the Abp118 bacteriocin, a bile salt hydrolase, a presumptive conjugation locus, and other genes potentially relevant for probiotic properties. Two subspecies of L. salivarius are recognized, salivarius and salicinius, and we detected megaplasmids in both subspecies by pulsed-field gel electrophoresis of sizes ranging from 100 kb to 380 kb. The discovery of megaplasmids of widely varying size in L. salivarius suggests a possible mechanism for genome expansion or contraction to adapt to different environments.

https://www.pnas.org/content/pnas/103/17/6718.full.pdf

Multireplicon genome architecture of Lactobacillus salivarius

Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry.

Florfenicol, a veterinary fluorinated analog of thiamphenicol, is approved for treatment of bovine respiratory pathogens in the United States. However, florfenicol resistance has recently emerged among veterinary Escherichia coli isolates incriminated in bovine diarrhea. The flo gene, which confers resistance to florfenicol and chloramphenicol, has previously been identified in Photobacterium piscicida and Salmonella enterica serovar Typhimurium DT104. The flo gene product is closely related to the CmlA protein identified in Pseudomonas aeruginosa. The cmlA gene confers nonenzymatic chloramphenicol resistance via an efflux mechanism. Forty-eight E. coli isolates recovered from calves with diarrhea, including 41 that were both chloramphenicol and florfenicol resistant, were assayed for the presence of both flo and cmlA genes. Forty-two of the 44 isolates for which florfenicol MICs were ≥16 μg/ml were positive via PCR for the flo gene. All E. coli isolates for which florfenicol MICs were ≤8 μg/ml were negative for the flo gene (n = 4). Twelve E. coli isolates were positive for cmlA, and chloramphenicol MICs for all 12 were ≥32 μg/ml. Additionally, eight isolates were positive for both flo and cmlA, and both florfenicol and chloramphenicol MICs for these isolates were ≥64 μg/ml. DNA sequence analysis of the E. coli flo gene demonstrated 98% identity to the published GenBank sequences of both serovar Typhimurium floSt and P. piscicida pp-flo. The flo gene was identified on high-molecular-weight plasmids of approximately 225 kb among the majority of florfenicol-resistant E. coli isolates. However, not all of the florfenicol-resistant E. coli isolates tested contained the large flo-positive plasmids. This suggests that several of the E. coli isolates may possess a chromosomal flo gene. The E. coli flo gene specifies nonenzymatic cross-resistance to both florfenicol and chloramphenicol, and its presence among bovine E. coli isolates of diverse genetic backgrounds indicates a distribution much wider than previously thought.

Characterization of Chloramphenicol and Florfenicol Resistance in Escherichia coli Associated with Bovine Diarrhea

A general method for detecting and sizing large plasmids.

Salmonella phage PSP3, another member of the P2-like phage group.

Rapid isolation of high-molecular-weight DNA from agarose gels.

IN constant conditions, the burst size of a particular bacteriophage is a characteristic of that bacteriophage. We have found aged P4/2 phage particles yield larger burst sizes than young phage particles.

/pdf/ageing-effect-on-phage-particles-leading-to-an-increase-in-4yyse5y5ez.pdf

Anthony J. Zuccarelli

Papers

A general method for detecting and sizing large plasmids.

Salmonella phage PSP3, another member of the P2-like phage group.

Rapid isolation of high-molecular-weight DNA from agarose gels.

Ageing Effect on Phage Particles leading to an Increase in Burst Size