Advanced Techniques in Diagnostic Microbiology

26. Molecular Strain Typing 453
and Brazilian lineages (Ip et al., 2003). The resistance profiles of MRSA clones
can vary widely; strain typing provides a method of distinguishing these clones
(Coombs et al., 2004).
MRSA is a clonal organism that is difficult to strain type with many common
genotypic techniques. PFGE is considered the gold standard for typing MRSA
strains; however, PFGE is a cumbersome technology to implement routinely in the
clinical laboratory. Many studies have been published that have tried to demonstrate
the level of PFGE strain discrimination with an easier or quicker technology. A
sequencing method that targets a polymorphic repeat region of the protein A gene,
called spa typing, has shown a high level of concordance to PFGE (Harmsen et al.,
2003; Faria et al., 2005); however, the discrimination was slightly lower for some
outbreaks (Tang et al., 2000). MLST may be used in concert with spa typing or
PFGE as a confirmation method (Perez-Roth, 2004; Faria, 2005). PCR methods
such as multiplex PCR with primers targeting MRSA-specific genes have not been
as discriminating as PFGE (Stranden et al., 2003). When manual rep-PCR was
compared with PFGE, one rep element showed excellent discrimination, but the
interlaboratory reproducibility was poor (Deplano et al., 2000). When automated
rep-PCR was compared with PFGE, however, the results were concordant (Shutt
et al., 2005). The reproducibility of automated rep-PCR using the DiversiLab
System has been established elsewhere (Healy et al., 2005).
Another common nosocomial pathogen is multidrug-resistant Acinetobacter
baumannii (IMRAB). As it is for MRSA, PFGE is a common typing technique for
Acinetobacter; however, decreased isolate clonality allows for successful typing
using simpler methods than PFGE. Many reports have established manual rep-
PCR as an accepted typing method of Acinetobacter (Liu and Wu, 1997; Bou
et al., 2000). Although slightly less discriminating than PFGE, the interpretation
is easier. Other methods include antibiotyping and RAPD (Mathai et al., 2001),
which appear to have less discrimination than rep-PCR (Martin-Lozano et al.,
2002).
Acinetobacter strain typing has been successfully used to track source infections
and outbreaks in health care facilities. Rep-PCR was used to determine a
definite source for multiple cases of bacteremia, and in a number of the cases, the
suspected source, originally determined through standard microbiological tests,
was refuted (Martin-Lozano et al., 2002). Typing by rep-PCR established that
another outbreak was transmitted with a humidifier temperature probe (Snelling
et al., 1996). Another study, using IRS-PCR typing, identified that a single strain
of multiresistant–A. baumannii was responsible for the prevalence of nosocomial
infection among surgical patients, clearly differentiating this outbreak from the
previous endemic situation (Wu and Della-Latta, 2002).
The incidence of infection from another vancomycin-resistant enterococcus
(VRE) has also grown in recent years (CDC, 2004). Due to its spread, the CDC
recommends typing isolates in facilities with continued or subsequent outbreaks
(CDC, 1995). Similar to MRSA, resistant strains of enterococcus are less diverse
than sensitive strains (Harrington et al., 2004). Strain typing of VRE is performed
using methods described in previous sections, but commonly by PFGE (Pfaller,