**4. Results**

Out of the total number of isolates, the largest number of isolates included in our research was isolated from the nasal or nasopharyngeal swab (41%), while 35% isolates were found in a skin swab samples. There was 1% isolates isolated from the umbilical swab and sputum sample, and 2% from an abscess puncture sample and conjuctival swab.

Based on the analysis of data from the epidemiological questionnaire, we estimated the distribution of examinees by gender. Out of a total of 100 subjects

#### *Infections and Sepsis Development*

included in the study, 49 (49%) were male and 51 (51%) female and there was no statistically significant difference in the percentage of the particular sex. The average age of the respondents was 10.9 ± 18.7 years.

Prior to the start of the study, 59% of subjects had hospital treatment according to the anamnestic data, but there was no statistically significant difference in the groups with and without hospital treatment in the year before the start of this study. Out of the 59 examinees who were hospitalized, 86.4% were hospitalized in the maternity hospital, while a significantly smaller number was hospitalized in orthopedics (5.1%), pediatrics (3.4%), dermatology, gynecology and surgery (1.7%).

Antibiotic usage within 12 months before the follow-up period was recorded in 32% of examinees, while 68% of them did not recieve therapy in that period, which is statistically significant. Out of the group of examinees who were taking antibiotics, 34.4% recieved amoxicillin and clavulanic acid, 18.8% recieved fourth-generation cephalosporins and sulfethoxazole-trimethoprim, while only 6.3% recieved macrolides.

MRSA was present in only 3% examinees out of those who had previosly documented MRSA strains (one year before this study). 6.0% of examinees included in the study had surgical procedure at the same time, while 94% of examinees didn't have surgery.

Epidemiological data are a significant variable for differentiating CA and HA MRSA. Therefore, variables that may have influenced the transmission and development of staphylococcal infections have been included in the epidemiological questionnaire. None of the subjects had been on dialysis therapy, nor had they been placed in a nursing home for 12 months prior to our study. 10% of examinees stated that they own a pet (80% own a dog as a pet and 20% a cat as a pet).

Out of total number of examinees, 8.0% of examinees had previous contact with a MRSA infected patients, 13% were involved in sports and 2.0% of examinees used an invasive /orthopedic device.

#### **4.1 Microbiological analysis of the strains**

During the follow-up period, out of a total of 4341 examined patient samples, 2279 *Staphylococcus aureus* were isolated, out of which 653 were methicillin-resistant SA. The prevalence of *Staphylococcus aureus* was 52.5% and the prevalence of MRSA was 28.7%.

We selected a representative sample of 100 strains, eliminating the "copy" strains (strains from the same patient at different places and those that are repeated), by random selection, trying to monitor the dynamics and even representation throughout the months of the study period).

#### **4.2 Results of antibiotic susceptibility testing**

In our study, 100 strains were tested for susceptibility to 12 antibiotics. Antibiotics that were tested include antibiotics that are therapeutically important for staphylococci, as well as other antibiotics that are known to be resistant to them as a marker for a virulent clone, such as fusidic acid.

All MRSA isolates were resistant to the β-lactam antibiotics tested, i.e. penicillin, oxacillin, and cefoxitin, 68% of MRSA strains were resistant to erythromycin, 5% to clindamycin, 5% to gentamicin and 4% to ciprofloxacin. All isolates were susceptible to sulphamethoxazole-trimethoprim, rifampicin, fusidic acid, linezolid and vancomycin (**Table 1**).

Based on the antibiogram, all examined strains were divided into 5 profile groups. Group A was represented in 32%, group B in 62%, group C 4%, group D 1% and 1% group E. (**Table 2**).

#### *Distribution and Molecular Detection of Methicilin-Resistant* Staphylococcus aureus *DOI: http://dx.doi.org/10.5772/intechopen.98655*


#### **Table 1.**

*Antimicrobial susceptibility patterns (N = 100).*


#### **Table 2.**

*Profile groups based on the antibiogram.*

#### **4.3 Results of phenotypic methods in MRSA detection**

In our study, conventional phenotypic methods were used to detect MRSA isolates: disk diffusion oxacilin test and disk diffusion cefoxitin test. To confirm the MRSA isolates, a latex agglutination test with antibodies to PBP2a, a selective chromogenic medium, ChromID MRSA, and an E test were used to determine the value of the minimum inhibitory concentration for oxacillin.

Oxacillin disk diffusion and cefoxitin disk diffusion tests, due to their accuracy and economic acceptability, have proven to be good options for the detection methicillin resistance of S.aureus. The results of this study indicate a previously proven fact that the latex agglutination test, ChromID MRSA, and E test, due to their high sensitivity and specificity, play a significant role as confirmatory tests for the detection of methicillin resistance.

All phenotypic methods that were used had 100% sensitivity as well as specificity, except for the DD cefoxitin and DD oxacillin tests, 98.9 and 96.8.

## **4.4 Molecular analysis of tested strains**

#### *4.4.1 MecA gene detection*

Reduced sensitivity or resistance to oxacillin or cefoxitin was found in all isolates, including the study by phenotypic methods. After detection of MRSA isolates by phenotypic methods, all isolates were subjected to molecular methods that tested the presence of the mecA gene.

All tested isolates were positive for the mecA gene. The control mecA positive strains used in our study are MRSA isolates described in the Methods section. One of the isolates used is COL and the other is WIS.

COL is an MRSA strain isolated for the first time in 1965 in Great Britain, known as a representative of the Archaic clone, whose characteristics are: SCCmec type I, ccr AB 1, sequential type (ST) 4.

WIS is an MRSA isolate native to Australia, SCCmec type V, possesses ccrC.

#### *4.4.2 Spa-typing test results*

As previously described in the Introduction to Spa Typing, the polymorphic region X consists of a variable number of 24 base pairs of repeating fragments. From a total of 100 strains examined, we selected a representative sample of 29 strains that underwent spa-typing. Seven different spa types were discovered: t008, t919, t041, t1179, t2187, t2674 and t10807. The most common type was t008 (55.2%).

#### *4.4.3 SCCmec typing*

We analysed and subjected 100 isolates to SCCmec typing. The obtained amplification products differ in molecular weight, and the typing result is obtained by visual comparison with the control strains. SCCmec loci of control strains were also amplified in each reaction cycle. A marker was applied to each individual gel, with a range of DNA fragments of 100-1500pb. The PCR products of the control strains were electrophoretically parallel separated. Each amplification cycle in which no corresponding PCR product was obtained in the control strains was repeated, as well as PCR reactions and electrophoresis of isolates that could not be typed with this method.

The distribution of SCCmec types is shown in **Figure 1**. The most prevalent SCCmec element was type IV (86%), followed by SCCmec I (4%) and SCCmec type

**Figure 1.** *SCCmec types distribution among MRSA isolates. Note: NT- non-typeable.*

*Distribution and Molecular Detection of Methicilin-Resistant* Staphylococcus aureus *DOI: http://dx.doi.org/10.5772/intechopen.98655*

V (1%). Non-typical strains were found in 9.0% of cases. No isolates with SCCmec III were found during this analysis. Chi-square test was used for the analysis of categorical variables.

#### *4.4.4 Determination of the presence of the PVL gene*

Using the primers and amplification cycle conditions described in the Method section, 100 isolates were tested for the presence of the PVL toxin gene. MW2 strain, MRSA strain isolated in 1998 in the USA, SCCmec type IV, ST131 was used as a positive control strain.

Positive expression of PVL gene was found in 24.0% of isolates, while 76% showed a statistically significant higher negative expression of PVL gene.

The current prevalence of CA MRSA PVL positive isolates was 0.05% and the periodic prevalence was 0.037%.

Common to all positive PVL MRSA isolates is SCCmec type IV and the fact that they are outpatient isolates.

#### *4.4.5 Results of PFGE analysis*

Results PFGE - electrophoretic profiles of isolates.

Pulsed field electrophoresis after the splitting of chromosomal DNA by the restriction enzyme SmaI yielded clearly separated DNA fragments that were visualized by UV light illumination after staining with ethidium bromide and photographed with a Polaroid camera.

**Figure 2** shows a photograph of electrophoretic gels that are part of the results obtained by this analysis. The number of fragments greater than 10 for each isolate is visible, the minimum of the bands to which the comparison standards described in the Methods section can be applied. **Figure 2** is a representation of a PFGE electrophoretic gel after cleavage with SmaI enzyme and separation in an electric field and staining with ethidium bromide.

M means a lambda marker, and genotyped strains are numbered 1–20. Blank fields 7 and 12 mean that the isolation probably failed in the first attempt and we repeated these isolates.

#### **Figure 2.**

*Representative PFGE patterns of MRSA strains isolated from population included in the study, after splitting enzyme SmaI and separation in an electric field and staining with ethidium bromide.*

#### *Infections and Sepsis Development*

Determination of PFGE groups using dendrogram percentage similarity:

The photos were scanned and stored in the database of the computer software system GelCompar. Each individual strip was subjected to a normalization procedure, in which, for each individual photograph, the size of the fragments was compared with the same molecular standard found on each gel in the electrophoretic reaction.

After normalization and entering basic data into the database, a dendrogram of similarity percentage was made, using Dice coefficient from PFGE data, using a limit value of ≥80%, with a tape tolerance of 3% and an optimization parameter of 0.5%.

In the PFGE profile analysis of the isolates with the computer system GelCompar, the isolates fell into five similarity groups: A-E. The largest number of isolates (87%) belonged to one of two groups: C (60%) and D (27%).

Group A includes 3 isolates, described as SCCmec type IV. There were no PVL positive isolates in this group.

Group B included four isolates, with three isolates being SCCmec type I, while one was SCCmec type IV. There were no PVL positive isolates in this group.

Group C represents the largest group in the PFGE analysis, including 60 isolates, and all isolates of this group belonging to SCCmec type IV. In this group, 28.33% (17) of isolates were PVL positive.

Group D includes 27 isolates, three of which were nontypeable, and the rest of the isolates belong to SCCmec type IV. In this group, only two isolates were PVL positive.

Group E included 3 isolates, SCCmec types I, IV and V. These three isolates were not PVL positive.

Three PFGE types were singletons, that is, not similar to any of the other strains, two of which are SCCmec IV, while one is nontypeable.
