Introduction:

A nosocomial infection (derived from the Greek words nosos [disease] and
komein [to care for], and later the Latin word for hospital nosocomium) is defined as an
infection that is not present or incubating when the patient is admitted to hospital or other
health-care facility (Garner et al., 1988). Hospital-acquired infections (HAI) increase
morbidity and mortality and constitute a high financial burden on health care systems
(Willemse-Erix et al., 2009). Vincent (2003) stated that the time frame for diagnosis of a
nosocomial infection will thus clearly be dependent on the incubation period of the
specific infection; 48–72 h after admission is generally deemed indicative of nosocomial,
rather than community acquired infection. Although generally associated with hospital
admission (hence the term hospital-acquired infection), nosocomial infections can arise
after admission to any health-care facility, and the term health-care associated infection is
increasingly being used. Such infections are common and associated with great morbidity
and mortality. Indeed, one provocative headline stated “Hospital acquired infections kill
5000 patients a year in England (Mayor, 2000).
Any organism can be implicated in nosocomial infection, and many infections are
polymicrobial (Vincent et al., 1995). Recent years have seen a swing in the pattern of
infecting organisms towards gram-positive infections (Friedman et al., 1998).The
surveillance and control of pathogens of epidemiologic importance project (SCOPE) data
(Edmond et al., 1999) revealed that gram-positive cocci were isolated in 64% of 10617
episodes of nosocomial bacteraemia, whereas gram-negative bacilli were isolated in only
27% of cases. The European Prospective Investigation into Cancer and Nutrition(EPIC)
study (Vincent et al, 1995) identified the following as the most commonly reported
nosocomial pathogens: Staphylococcus aureus (30%), Pseudomonas aeruginosa (29%),
coagulase-negative staphylococci (19%), yeasts (17%), Escherichia coli (13%),
enterococci (12%), Acinetobacter spp. (9%), and Klebsiella spp. (8%) (Spencer et al.,
1996). Other studies have noted similar patterns of causative microorganisms (Richards et
al., 2000).
An effective weapon against HAI is early detection of potential outbreaks and
sources of contamination (Willemse-Erix et al., 2009).

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Typing of nosocomial pathogens includes:
I. Phenotyping methods.
II. Molecular methods.
I. Phenotyping methods:
These methods examine the physical attributes, biochemical products, and
chemical requirements of microorganisms (Leung et al., 2004; Environmental
Protection Agency EPA, 2005). Antimicrobial susceptibility testing is a common practice
in the clinical microbiology laboratory. The resultant antibiogram indicates the pattern of
in vitro resistance or susceptibility of an organism to a panel of antimicrobial agents
(Barenfanger et al., 1999). Serotyping uses a series of antibodies to detect antigens on
the surface of bacteria that have been shown to demonstrate antigenic variability (Ko et
al., 2000; Babl et al., 2001). Bacteriophage and bacteriocin typing as epidemiologic tools
are limited to bacteria. Bacteriophage (phage) typing classifies bacteria based on the
pattern of resistance or susceptibility to a certain set of phages (Hopkins et al., 2004).

II. Molecular methods:

Strommenger et al. (2008) issued that the use of efficient and accurate
epidemiological typing methods is a prerequisite for monitoring and for limiting the
occurrence and spread of epidemic clones within and between hospitals. Identification by
molecular methods allows for more rapid and accurate identification of etiologic agents
in a much shorter time than traditional methods. For example, a protocol using real-time
PCR to detect and differentiate Gram-positive from Gram-negative bacteria could yield
results in less than 3 hours, inclusive of preparation time (Klaschik et al., 2002). Such
rapid identification would allow for the earlier initiation of a focused antimicrobial
regimen, and decrease the likelihood of disease progression (Doern et al., 1994).
Therefore, the use of strain typing in infection control decisions is based on
several assumptions: (i) isolates associated with the outbreak are recent progeny of a
single (common) precursor or clone, (ii) such isolates will have the same genotype, and
(iii) epidemiologically unrelated isolates will have different genotypes (Singh et al.,
2006).

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 Many microbial typing methods are available: PCR multilocus enzyme
electrophoresis (MLEE), multilocus sequence typing (MLST), pulsed-field gel
electrophoresis (PFGE), restriction fragment length polymorphisms (RFLP), DNA
sequencing, ribotyping, restriction fragment length polymorphism studies, randomly
amplified polymorphism DNA (RAPD), amplified fragment length polymorphism
(AFLP) and repetitive sequence-based PCR (REP-PCR) (Healy et al., 2005).
Typing Methods Using Polymerase chain reaction (PCR)
During the past decade, advances in PCR technology and other DNA signal and
target amplification techniques have resulted in these molecular diagnostics becoming
key procedures (Wagar, 1996). Such techniques are conceptually simple, highly specific,
sensitive, and amenable to full automation (Klapper et al., 1998).
Multiplex PCR
In multiplex PCR more than one target sequence can be amplified by including
more than one pair of primers in the reaction and it has the potential to produce
considerable savings of time and effort within the laboratory without compromising test
utility (Elnifro et al., 2000). Since its introduction, multiplex PCR has been successfully
applied in many areas of nucleic acid diagnostics, including gene deletion analysis
(Chamberlain et al., 1989), mutation and polymorphism analysis (Rithidech et al., 1997),
quantitative analysis (Sherlock et al., 1998), and RNA detection (Zou et al., 1998).
The multiplex PCR assay offers a rapid, simple, and accurate identification of
antibiotic resistance profiles and could be used in clinical diagnosis as well as for the
surveillance of the spread of antibiotic resistance determinants in epidemiological studies
(Strommenger et al., 2003).
Nested PCR
Nested PCR involves the sequential use of two PCR primer sets. The first primer
set is used to amplify a target sequence (which increases the sensitivity for the second
primer set); the amplicon generated then serves as the template for a second amplification
using primers internal to those of the first amplicon (Singh et al., 2006). Ginevra et al.
(2009) found that in Legionnaires' disease nested PCR-based SBT (NPSBT) applied
directly to clinical specimens improved significantly epidemiological typing as compared
to the initial Sequence-based typing (SBT) in particular when no isolates are available.
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Arbitrarily primed PCR
Arbitrarily primed PCR (AP-PCR) and the very similar randomly amplified
polymorphic DNA assay are variations of the PCR technique in which a random primer,
which is not targeted to amplify any specific bacterial DNA sequence, is used for
amplification (Eribe et al., 2000). Grattard et al. (1994) found that AP- PCR was a very
discriminatory tools for the investigation of nosocomial outbreaks caused by
Enterobacter cloacae. The DNA polymorphism assay most recently introduced for the
typing of Group A streptococcus (GAS; Streptococcus pyogenes) is random amplified
polymorphic DNA (RAPD) analysis (Welsh et al., 1990), which has also been called
arbitrarily primed PCR.
Multilocus enzyme electrophoresis (MLEE)
The application of multilocus enzyme electrophoresis (MLEE) to meningococcal
isolate collections (Selander et al., 1986) has established the existence of particular
meningococcal lineages associated with invasive disease and played a seminal role in
elucidating the epidemiology and population biology of the meningococcus (Caugant et
al., 1986); however, for largely practical reasons this technique has not been widely
adopted by reference laboratories and has rarely been employed during outbreak
investigations (Tzanakaki et al., 2001).
Repetitive sequence-based PCR (REP-PCR)
An alternative approach to PCR-based fingerprinting, repetitive-element PCR
(rep-PCR), uses as primers oligonucleotides homologous to defined sequences which are
present in multiple copies in the bacterial genome (Versalovic et al., 1994). Indeed, rep-
PCR's same-day reproducibility and discriminating power have sufficed for small-scale
epidemiological and phylogenetic studies involving wild-type E. coli strains (Johnson et
al., 1998).

Pulsed-field gel electrophoresis (PFGE)

Pulsed-field gel electrophoresis has been used effectively as a molecular
subtyping tool in outbreak investigations (CDC, 2001) and surveillance (Swaminathan et

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al., 2001). Faria et al. (2008) stated that pulsed-field gel electrophoresis (PFGE), remains
the method of choice in many laboratories due to the extensive experience with this
methodology and the large body of data accumulated using the technique. Pulsed-field gel
electrophoresis (PFGE) has been accepted as the "gold standard" for molecular strain
typing of Methicillin-resistant Staphylococcus aureus (MRSA) (Blanc et al., 2001).
Although the discriminatory power of PFGE has compared favorably to that of other
subtyping methods (Hopkins et al., 2000), the similarity of PFGE patterns has not been
stringently evaluated as a measure of genetic relatedness. This is most critical for
transmission studies, which rely on molecular genetic data for making inferences about
routes of pathogen spread (Chasseignaux et al., 2001; Midgley et al., 2001).
Restriction fragment length polymorphism (RFLP)
The standardized IS6110 restriction fragment length polymorphism (RFLP)
typing of Mycobacterium tuberculosis isolates (Van Embden et al., 1993) is based on the
concept that RFLP patterns reflect the presence of the IS6110 element at different sites in
the genome of M. tuberculosis complex strains (Kremer et al., 1999). RFLP typing has
permitted the differentiation of clinical M. tuberculosis isolates in many parts of the world
(Horgen et al., 1998).
Southern Blot Analysis-Ribotyping
One of the most common targets for Southern blotting is the gene for the rRNA,
and the targeting of the rRNA gene is referred to as ribotyping (Singh et al., 2006).
Typically, the discriminatory power of ribotyping has been shown to be less that of PFGE
or some PCR-based (Bailey et al., 2002).
Brisse et al. (2002) included that automated ribotyping appears to be a very
valuable approach for characterizing Vancomycin-resistant Enterococcus faecium
(VREF) strains.
Amplified fragment length polymorphism (AFLP)
AFLP is a genotypic library-based method that can produce strain-specific
fingerprints for closely related bacteria (Geornaras et al., 2001; Leung et al., 2004).
AFLP is very similar to the RFLP (Restriction Fragment Length Polymorphism) analysis
and consists of three basic steps: 1) digestion of DNA with restriction enzymes and
ligation of specific adaptors to the restriction fragments; 2) preamplification and selective
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amplification of the fragments with corresponding primers; and 3) electrophoretic
separation of the products on a high resolution gel (Janssen et al., 1996; Liscum and
Oeller, 2006).
Selective amplification of ApaI/TaqI templates with primer combination A02-T02
(both having an additional C at their 3' end) generated auto radiographic AFLP
fingerprints that were grouped by numerical analysis in two main AFLP clusters allowing
clear separation of M. ulcerans (cluster I) from the M. tuberculosis complex members
M. bovis and M. tuberculosis (cluster II) (Huys et al., 2000).
Plasmid Analysis:
Plasmid typing was the first molecular method to be used as a bacterial typing
tool (Archer et al.,1984). Typing is performed through the isolation of plasmid DNA and
comparison of the numbers and sizes of the plasmids by agarose gel electrophoresis
(Singh et al., 2006). Plasmid analysis has been applied in clinical situations to determine
the evolution and spread of antibiotic resistance among isolates with different PFGE
profiles or among different species of organisms within hospitals (Donabedian et al.,
2003; Feil et al., 2003). Singh et al. (1992) had found a high rate of fecal colonization
with trimethoprim resistance (Tmpr) Escherichia coli and, using total plasmid content
analysis, had shown that this was due to a diversity of strains.

RECENT ADVANCES IN MOLECULAR TYPING: NUCLEOTIDE

SEQUENCE-BASED ANALYSIS

Sequence-based molecular epidemiology is attractive in offering the promise of

reproducible typing profiles that are highly amenable to standardization, uniform
interpretation, and database cataloging, since they are based on simple quaternary data
(A, T, G, and C) (Kemp et al., 2005).

Single-locus sequence typing (SLST)

Sequence data for specific loci (genes for virulence, pathogenicity, drug
resistance, etc.) from different strains of the same species have revealed variability in a

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specific gene (Singh et al., 2006). At present, the single-locus sequence typing (SLST)
approach with most promise involves analysis of a particular region of the staphylococcal
protein A gene (spa) which is polymorphic due to 24-bp repeat sequences that may vary
in both the number of repeats and the overall sequence in the polymorphic X or short
sequence repeat region (Koreen et al., 2004).

Multi-locus sequence typing (MLST)

MLST was designed based on the principles of multilocus enzyme electrophoresis
(MLEE) in which the electrophoretic mobilities of housekeeping enzymes of isolates of
interest are compared (Enright et al., 1999). Based on the resulting migration pattern of
each enzyme, an allele number is assigned for each housekeeping locus. Once arranged
into a string of integers, the allele numbers at each locus define the electrophoretic type
(ET) of a strain (Selander et al., 1980). Being sequence-based, MLST provides a
definitive characterization of bacterial isolates that is consistent from one laboratory to the
next. The nuclei acid sequences are typically stored in a public database that can be
readily accessed via the Internet (González-Escalona et al., 2008).
Recently, a method based on the unique lengths of the intergenic regions
containing repetitive DNA loci (Tenover et al., 2007; van Belkum, 2007), known as
multiple-locus variable-number tandem-repeat analysis (MLVA), was introduced and
described.