Heteroresistance to vancomycin and novel point mutations in Tn1546 of Enterococcus faecium ATCC 51559.

A clinical strain of Enterococcus faecium ATCC 51559 exhibits heteroresistance, i.e. a high level of resistance to vancomycin (minimum inhibitory concentration (MIC)>256 microg/mL) by broth dilution but sensitivity to vancomycin by Etest (MIC=1.8 microg/mL). Three variants of this strain, EF1, EF2 and EF3, exhibit high levels of resistance to vancomycin both by broth dilution and Etest assays. The four strains were used to study heteroresistance by pulsed-field gel electrophoresis (PFGE), polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequence analysis of a partial region of the van operon. Minor differences between SalI and SmaI restriction profiles of the variants and the parental strain were observed by PFGE analysis. PCR analysis confirmed the presence of the vancomycin resistance marker vanA (0.73 kb) and a larger than expected amplicon (8.2 kb vs. 6.7 kb) of the van operon in all the strains. The 8.2kb van operon was cloned for EcoRI RFLP and sequence analysis. All of the clones exhibited distinctly different RFLP profiles when grown in the presence of kanamycin or vancomycin+kanamycin. The presence of these antibiotics during overnight growth of EF1 on plates also resulted in altered SalI PFGE profiles. Sequence analysis of the van operon clones revealed a 1.5kb IS1251-like insertion element between the vanS and vanH genes in all the strains. Several novel point mutations in the vanR, vanS, vanH, vanA, vanX and vanY genes were also discovered. Some of these mutations were present in the parental strain only and included base substitutions T-->C, A-->G, T-->A and T-->C at nucleotide positions 4202, 4597, 4763 and 6207 of Tn1546, resulting in amino acid replacements I76-->T and K208-->E of vanR, S19-->T of vanS and L64-->P of vanH genes, respectively. We believe that these are responsible for the observed heteroresistance. The present study clearly shows how independent novel mutations can give rise to polymorphism, heteroresistance and clonal diversity among vancomycin-resistant enterococci strains as a result of continuous exposure to antibiotics.

Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome.

BACKGROUND: A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome. RESULTS: The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the S. enteritidis genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2) of Influenza A virus, a domain of CD154 (CD154s) or a combination of both into the outer membrane protein LamB of S. enteritidis. CONCLUSION: We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed.