Antibiotic Resistance Protocols (Methods in Molecular Biology)

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the host organism except for the introduction of a resistance

cassette, consider carefully the orientation of the plasmid-

borne genes and their effects on surrounding loci once inserted

into the host. For example, it is recommended to integrate the

plasmid downstream of an operon that can be easily assayed for

disruption (e.g., the terminal gene in an amino acid biosynthe-

sis pathway): once constructed, the S. aureus mutants can be

screened and any auxotrophs discarded.

2. You may use either purified genomic DNA or a fresh bacterial
   colony as the template for this amplification. Note that some
   high-fidelity enzymes cannot tolerate a high concentration of
   cellular debris, so for colony PCR, dilution of a colony in
   100 μL water (then using 1 μL of this as the final reaction tem-
   plate) is recommended.


3. The suicide vector to be used should replicate in E. coli but not
   S. aureus, such that it can be manipulated easily but then inte-
   grates into the chromosome upon transformation into the lat-
   ter species. pMUTIN4 is a good example of a compatible
   suicide vector for integration into gram-positive organisms.
   Alternatively, it may be possible to use a temperature-sensitive
   replicon.


4. Colony PCR can be used to analyze transformants. It is recom-
   mended that plasmids passing this initial screening step are
   subsequently sequenced to confirm the absence of point
   mutations.


5. This step should take approximately 1 h. Check culture turbid-
   ity after 30 min and discard if growth takes longer than 90 min.


6. If transforming a suicide vector, the competent cell preparation
   described should be used immediately. If using this protocol
   for transformation of a shuttle or temperature-sensitive vector,
   aliquots may be frozen at − 80 °C for long term storage and
   gently defrosted prior to use.

Fig. 2 Output of a three-strain infection study. Each ring shows the strains isolated from a single animal. The
number within each ring gives log 10 (total CFU), i.e., the overall bacterial load. (a) Three strains represented
approximately equally. (b) Two dominant strains; the third is missing. (c) One dominant strain, but the low CFU
total implies that the viable counts were close to the detection limit. Care should be taken when extrapolating
strain ratios from low-CFU data

Construction and Use of Staphylococcus aureus Strains to Study Within-Host...