**6. Epidemiology and MLST**

100 Epidemiology Insights

Recently, Zhang et al. (2009) have described a new SCC*mec* type which has been denominated as SCC*mec* VIII and was found in an MRSA sample from a hospital. This new cassette has a size of 32.168 base pairs, and genes *mecA mecR1 mecI* of the *mec* complex and *ccrA4 ccrB4* of the *ccr* complex are present in addition to transposon Tn554, which is also

In addition to the characterization of SCC*mec* types as an epidemiological tool, other methods are available for molecular epidemiology studies, such as Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) and *spa* typing, which allow for the identification of circulating bacterial clones in hospitals and in the community.

PFGE is a method that uses restriction enzymes to cleave the extracted DNA, and in the case of the *Staphylococcus* genus, the enzyme used is *Sma*I. The fragments are separated in agarose gel. The direction of the electrical field is periodically changed, forming a 120º angle. This allows for fragments of up to 12mb to be separated, contrarily to conventional electrophoresis, which is not capable of separating fragments that are larger than 50kb (Herschleb et al., 2007). This method is generally used in local studies, such as in hospital

The sequencing of constitutive bacterial genes can also be used as an important epidemiological tool. The method known as *spa* typing investigates polymorphisms on a single locus. It can discriminate between PFGE and MLST and is also applied in local studies

Another methodology that uses the sequencing and analysis of genetic polymorphisms is MLST. It analyzes the sequence of seven constitutive genes (*gmk, pta, dfp, gtr, mutS, pyrR,*  and *xpt*) and compares them with the sequences of each allele in the locus, which are previously numbered. The combination of the alleles identified in each gene determines the profile of the sample. (Aanensen & Spratt, 2005). It is the most adequate methodology for detecting pandemic clones given that the investigated genes are constitutive, with low mutation rates when compared to the analysis of the whole chromosome, in the case of

Currently, *Staphylococcus spp*. samples have been typed in numerous epidemiological studies by means of the methods described above. By using these tools, 05 large pandemic clones have been described. These are the Brazilian clone, characterized by *SCCmec* IIIA, MLST 2-3- 1-4-4-3 (ST 239); the Iberian clone with type-IA *SCCmec*, and MLST 3-3-1-1/12-4-4-16 (ST 247); the clone known as New York / Japan, with type-III SCC*mec*, pattern MLST 1-4-1-4-12- 1-10 (ST 05); the Hungarian clone, with SCC*mec III*, MLST 2-3-1-1-4-4-3 (ST 239); the pediatric clone, with type-IV SCC*mec*, MLST 1-4-1-4-12-1-10 (ST 05) and the last large clone known as EMRSA-16, *SCCmec* II MLST 15-12-16-02-16-02-25-17-24-24-24 (ST 36) (Oliveira et al., 2002; Melter et al., 2003; Velásquez-Meza et al. 2004; Aires de Souza & De Lencastre,

In our scenario, the predominant MRSA samples belong to the Brazilian clone (Oliveira et al., 2001). The first report on the emergence of this clone dates to 1992-93 in various hospitals

outbreaks, which allows for identifying the circulating type.

and for detecting pandemic clones (Malachowa et al., 2005).

present in type II.

**4. Molecular epidemiology** 

PFGE (Miragaia et al., 2007).

2004, Aires Ribeiro et al., 2005).

**5. Pandemic clones of MRSA samples** 

The MLST method was developed by Maiden et al. (1998) by the sequencing and analysis of the loci of eleven constitutive genes of *Neisseria meningitidis*, and it is presently applied in molecular epidemiology studies on various pathogens, among which are *S. aureus* and *S. epidermidis*.

This methodology has been used in numerous studies on molecular epidemiology with good results due its good reproducibility, thus allowing for the detection of pandemic clones from circulating *S. aureus* and *S. epidermidis* samples.

Geng et al. (2010) reported the presence of clone ST59-MRSA-IV in China in community samples isolated from 47 children with impetigo and abscesses. In Malaysia, a study on 36 samples isolated from a hospital in Klang Valley for a five-month period reported a rate of 83.3% of samples belonging to clone ST 239 *SCCmec* III (Neela et al., 2010).

MLST has been used for studies in which circulating clones are identified in replacement of another already established clone. Sola et al. (2006) reported the emergence of a new clone identified as ST5 in hospitals in Cordoba, Argentina, in replacement of the Brazilian clone (ST239), which circulates in that region. In that study, 103 MRSA samples isolated from April to June 2001 and 31 MSSA samples isolated from 1999 to 2002 were used.

Epidemiological Aspects of Oxacillin-Resistant

*Staphylococcus aureus* (VRSA).

Source: CDC (2010)

*Staphylococcus* spp.: The Use of Molecular Tools with Emphasis on MLST 103

for individual patients if resistant infections are not quickly identified and the patient continues the ineffective treatment. Vancomycin-resistant Enterococcus (VRE) emerged in 1987 and the transfer potential of such resistance to other bacteria, vancomycin resistance surveillance has been an object of great scientific interest worldwide. Vancomycin resistance emerged in more common pathogenic organisms during the 1990s and 2000s, including vancomycin-intermediate *Staphylococcus aureus* (VISA) and vancomycin-resistant

In 1996, the first clinical *S. aureus* isolate with reduced vancomycin sensitivity, with MIC value in the intermediate range (MIC = 8 g/ml) and referred to as VISA was reported in Japan (Hiramatsu et al. 1997). Additionally, in June 2002 [44], eight patients with infections caused by *S. aureus* with reduced vancomycin sensitivity were confirmed in the United States. One month later, the Centers for Disease Control and Prevention (CDC) published the first reported on vancomycin-resistant *S. aureus* (VRSA, with MIC = or 32 g/ml) in a patient in Michigan, United States. The sample isolated from the patient contained the *vanA* gene as well as the *mecA* gene for oxacillin resistance. The presence of the *vanA* gene in this VRSA suggests that resistance may have been acquired through the passage of genetic material from vancomycin-resistant enterococci to *S. aureus.* In October of the same year [44], the second clinical isolate of VRSA was reported in a patient in Pennsylvania. The VRSA isolate also contained the *vanA* and the *mecA* genes. The presence of the *vanA*  gene suggests that the resistance determinant was acquired from vancomycin-resistant *Enterococcus* isolated from the same patient. April 2004, the third VRSA isolated from a patient in New York was reported. The isolate also contained the oxacillin- and vancomycinresistance *mecA* and *vanA* genes, respectively. According to CDC, the three VRSA isolated did not seem to be epidemiologically related (CDC 2002; CDC 2004). The CDC (2010) has recently confirmed the 11th case of vancomycin resistant *Staphylococcus aureus* (VRSA) infection since 2002 in the United States (Table 2). This serves as a reminder about the

> **Case State Year Age Source** 1 Michigan 2002 40 Plantar ulcers &

2 Pennsylvania 2002 70 Plantar ulcer 3 New York 2004 63 Urine from a

4 Michigan 2005 78 Toe wound 5 Michigan 2005 58 Surgical site wound

6 Michigan 2005 48 Plantar ulcer 7 Michigan 2006 43 Triceps wound 8 Michigan 2007 48 Toe wound 9 Michigan 2007 54 Surgical site wound

10 Michigan 2009 53 Plantar foot wound 11 Delaware 2010 64 Wound drainage

Table 2. Vancomycin resistant *Staphylococcus aureus* (VRSA) infection in the United States

Catheter tip

nephrostomy tube

after panniculectomy

after foot amputation

Previous studies have used MLST for comparison between samples and the prevalence between different pandemic clones. Campanille et al. (2009) analyzed 301 MRSA samples isolated from 19 Italian hospitals from 1990 to 2001. An increase of clone ST228, known as Italian clone, was observed from 2000 to 2007, conjointly with the decrease of clone ST247 (Iberian clone) when compared to the 1990-1999 period.

Clone ST228 is also associated with patients with cystic fibrosis. A study conducted on 93 MRSA samples isolated from patients with that disease at a treatment center in Madrid, Spain, identified 15 different PFGE patterns. A sample of each of these patterns was typed by MLST, with clone ST228 showing higher prevalence, with eight pulsetypes, followed by ST5, with two pulsetypes, and ST247, ST72 and ST255 with one pulsetype each (Molina et al., 2008).

In addition to epidemiological studies in hospitals and the detection of these pandemic clones from MRSA samples, the MLST method has been used to detect the transmission of oxacillin-resistant samples from animal reservoirs to humans, including its detection in hospitals. Some studied have detected the presence of MRSA clones in swines, thus emphasizing the importance of such animals as reservoirs. Smith et al. (2009) reported the presence of ST398 samples in swines and farmers in the mid-western region of the United States, which suggests transmission between animals and their breeders. In Germany, 1,600 swabs from swines from 40 farms were analyzed in a study where samples typed as ST398 were also identified (Köck et al., 2009).

MLST is usually used for detecting pandemic clones with good results. But this method can also be utilized for characterizing samples involved in hospital outbreaks.

Peacock et al. (2002) compared the MLST and PFGE methods in a study conducted on 104 *S. aureus* samples isolated from nasal swabs from patients under renal therapy. The isolated samples were typed by the MLST and PFGE methods with a similar discriminatory power between the two techniques for identification of circulating samples in hospitals.

Vindel et al. (2009) analyzed 463 *S.aureus* samples isolated from 145 Spanish hospitals in 2006. In addition to MLST, several methodologies such as PFGE, spa typing, *SCCmec*  characterization and *agr* typing were used. MLST showed good correlation with the methodologies used for detecting circulating samples, with applicability in localized nosocomial studies.

The combination of the two molecular epidemiology techniques can increase discriminatory power for analysis of different clones. Cookson et al. (2007) reported that the combination of the MLST and *SCCmec* methods are more appropriate for multi-center studies. The authors analyzed MRSA samples from various European countries by means of PFGE, MLST, *SCCmec* analysis and *spa* typing.
