**4. Methodology**

To identify the most appropriate scales for measuring the relationships proposed with a high degree of reliability, validity, and dimensionality, it developed a process with several stages [51]. The first stage consisted of constructing scales with a good degree of content validity. It made an exhaustive literature review, taking as reference the scales constructed in various prior studies of affective evaluation [52, 53], symbolic benefit [54, 55], functional benefit [56], hedonic benefit [56, 57], and satisfaction [58–60].

A study of critical incidents was done, in which people were asked to describe the factors that formed part of the constructs analyzed. Fifty people chosen by nonprobabilistic convenience sampling participated in this study, from which was obtained the prior scales of the study constructs. A second procedure was done to purify these scales, following the recommendations of [61]. There was also a series of "focus groups" composed of regular clients from different banks in Chile, as well as interviews with business experts and executives from the banking industry. These analyses permitted us to identify the indicators that best reflect the dimensions needed for this study, reformulating and/or eliminating indicators that were problematic or repetitive using a modification of the [62]. Each expert was asked to classify items relative to their dimension, considering three alternatives: clearly, somewhat, or not at all representative. Finally, it was decided to preserve the items in which there was a high level of consensus [63]. From these analyses, it obtained the scales that enabled us to develop the questionnaire. In the second stage, after constructing the questionnaire, it performed a quantitative pre-test with a random sampling of 50 people. Using these data, it calculated an exploratory factor analysis and the Alpha Cronbach for each of the resulting dimensions. This initial analysis enabled us to confirm the existence of each dimension resulting from the preceding analyses. The items were written as statements (see **Table 1**) to be evaluated using a 7-point Likert scale.

It used nonprobabilistic quota sampling proportional to the market participation of the first three leading banks in the private banking sector in Chile: Santander Bank (18%); Chile Bank (17%), and BCI Bank (15%)1 . The surveys were administered outside the main branches of each bank, especially in the downtown of Santiago.

that did not adhere well to their dimension, it made various principal components factor analyses with Varimax rotation [64]. This procedure did not indicate the need to eliminate any indicator from the scales analyzed (see **Table 2**); all scales showed a good degree of one-

Taking into account all of the scales constructed, it developed a CFA using structural equations modeling (SEM) to confirm whether the indicators or variables were appropriate for achieving good model fit. It used the three criteria proposed by [66]. The first criterion consists of eliminating indicators that had a weak convergence condition with their corresponding latent variable. It took as limit a t-student value higher than 2.28 (p = 0.01). The second criterion consisted of eliminating from the analysis variables whose loadings translated into standardized coefficients lower than 0.5. Finally, it also had to eliminate the indicators that showed a linear relationship

22. Following this analysis, it was not necessary to eliminate any indicator. The fit indices for the confirmatory factor analysis were acceptable: IFI = 0.948, CFI = 0.948, RMSEA = 0.078, Normed χ2 = 5.81. Once verified the optimal model, it confirmed the reliability of each scale by applying three tests: Alpha Cronbach (limit = 0.7), composite reliability of the construct (limit = 0.7) [67],

lower than 0.3. In this process, it used the statistical package AMOS SPSS

Antecedents of Satisfaction with Financial Services: Role of Perceived Benefits

http://dx.doi.org/10.5772/intechopen.74653

101

dimensionality, with factor loadings well over 0.4 [65].

with goodness of fit, R2

**Table 1.** Measurement scales.

In the third stage, the data was collected. The survey was applied to a total sample of 850 people. It has, however, to eliminate 64 forms because they were incomplete and/or incorrectly answered, leaving a definitive sample of 786 people. For the responses, the last visit the bank was taken as a reference at which the respondents were customers. Of the total interviewees, 64% were men and 49% were married (83% were 25–54 years old, 84% held university degrees, 89% worked full time, and 81% had monthly incomes over 750,000 Chilean pesos (1.114 USD)2 .

With the data obtained, it has been made a psychometric analysis to develop scales with a good degree of reliability, validity, and dimensionality. It applied exploratory factor analysis, confirmatory factor analysis (CFA), and various reliability analyses with the Alpha Cronbach statistics, reliability construct, and average variance extracted (AVE). To identify the items

1 Risk rating ICR. Knowledge & Trust, December 2016.

2 Exchange rate december 23, 2016.

**Table 1.** Measurement scales.

**4. Methodology**

100 Marketing

hedonic benefit [56, 57], and satisfaction [58–60].

statements (see **Table 1**) to be evaluated using a 7-point Likert scale.

each bank, especially in the downtown of Santiago.

Risk rating ICR. Knowledge & Trust, December 2016.

Exchange rate december 23, 2016.

(17%), and BCI Bank (15%)1

1

2

To identify the most appropriate scales for measuring the relationships proposed with a high degree of reliability, validity, and dimensionality, it developed a process with several stages [51]. The first stage consisted of constructing scales with a good degree of content validity. It made an exhaustive literature review, taking as reference the scales constructed in various prior studies of affective evaluation [52, 53], symbolic benefit [54, 55], functional benefit [56],

A study of critical incidents was done, in which people were asked to describe the factors that formed part of the constructs analyzed. Fifty people chosen by nonprobabilistic convenience sampling participated in this study, from which was obtained the prior scales of the study constructs. A second procedure was done to purify these scales, following the recommendations of [61]. There was also a series of "focus groups" composed of regular clients from different banks in Chile, as well as interviews with business experts and executives from the banking industry. These analyses permitted us to identify the indicators that best reflect the dimensions needed for this study, reformulating and/or eliminating indicators that were problematic or repetitive using a modification of the [62]. Each expert was asked to classify items relative to their dimension, considering three alternatives: clearly, somewhat, or not at all representative. Finally, it was decided to preserve the items in which there was a high level of consensus [63]. From these analyses, it obtained the scales that enabled us to develop the questionnaire. In the second stage, after constructing the questionnaire, it performed a quantitative pre-test with a random sampling of 50 people. Using these data, it calculated an exploratory factor analysis and the Alpha Cronbach for each of the resulting dimensions. This initial analysis enabled us to confirm the existence of each dimension resulting from the preceding analyses. The items were written as

It used nonprobabilistic quota sampling proportional to the market participation of the first three leading banks in the private banking sector in Chile: Santander Bank (18%); Chile Bank

In the third stage, the data was collected. The survey was applied to a total sample of 850 people. It has, however, to eliminate 64 forms because they were incomplete and/or incorrectly answered, leaving a definitive sample of 786 people. For the responses, the last visit the bank was taken as a reference at which the respondents were customers. Of the total interviewees, 64% were men and 49% were married (83% were 25–54 years old, 84% held university degrees, 89% worked full time, and 81% had monthly incomes over 750,000 Chilean pesos (1.114 USD)2

With the data obtained, it has been made a psychometric analysis to develop scales with a good degree of reliability, validity, and dimensionality. It applied exploratory factor analysis, confirmatory factor analysis (CFA), and various reliability analyses with the Alpha Cronbach statistics, reliability construct, and average variance extracted (AVE). To identify the items

. The surveys were administered outside the main branches of

.

that did not adhere well to their dimension, it made various principal components factor analyses with Varimax rotation [64]. This procedure did not indicate the need to eliminate any indicator from the scales analyzed (see **Table 2**); all scales showed a good degree of onedimensionality, with factor loadings well over 0.4 [65].

Taking into account all of the scales constructed, it developed a CFA using structural equations modeling (SEM) to confirm whether the indicators or variables were appropriate for achieving good model fit. It used the three criteria proposed by [66]. The first criterion consists of eliminating indicators that had a weak convergence condition with their corresponding latent variable. It took as limit a t-student value higher than 2.28 (p = 0.01). The second criterion consisted of eliminating from the analysis variables whose loadings translated into standardized coefficients lower than 0.5. Finally, it also had to eliminate the indicators that showed a linear relationship with goodness of fit, R2 lower than 0.3. In this process, it used the statistical package AMOS SPSS 22. Following this analysis, it was not necessary to eliminate any indicator. The fit indices for the confirmatory factor analysis were acceptable: IFI = 0.948, CFI = 0.948, RMSEA = 0.078, Normed χ2 = 5.81. Once verified the optimal model, it confirmed the reliability of each scale by applying three tests: Alpha Cronbach (limit = 0.7), composite reliability of the construct (limit = 0.7) [67],

**Table 2.** Exploratory factorial analysis of the scales.

and analysis of the variance extracted (limit = 0.5) [68]. The results show that the minimum values established for these reliability parameters are fulfilled in all cases (see **Table 3**).

**Table 3.** Reliability analysis of scales.

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**Table 4.** Discriminant validity.

The validity of the SEM model was confirmed using content and construct validity. The scales show a good degree of content validity, since it made a thorough analysis of the literature, a study of critical incidents with customers, and then a purification of the scale using different "focus groups" with customers and in-depth interviews with commercial experts and executives from different banks. To fulfill construct validity, it analyzed if the proposal, purified scale fulfilled the conditions for convergent and discriminant validity. Convergent validity was confirmed by observing that all of the standardized coefficients of the CFA were statistically significant at 0.01 and higher than 0.5 [69]. To confirm the presence of discriminant validity, it was used the confidence interval test [70], which consists of constructing the confidence intervals resulting from the correlations between the different latent variables that compose the CFA model (see **Table 4**). This test indicates the model's discriminant validity, since no confidence interval contained the value of 1 [71].

With all of these antecedents, it concluded that the proposes model shows a good degree of general validity.

**Table 3.** Reliability analysis of scales.

and analysis of the variance extracted (limit = 0.5) [68]. The results show that the minimum val-

The validity of the SEM model was confirmed using content and construct validity. The scales show a good degree of content validity, since it made a thorough analysis of the literature, a study of critical incidents with customers, and then a purification of the scale using different "focus groups" with customers and in-depth interviews with commercial experts and executives from different banks. To fulfill construct validity, it analyzed if the proposal, purified scale fulfilled the conditions for convergent and discriminant validity. Convergent validity was confirmed by observing that all of the standardized coefficients of the CFA were statistically significant at 0.01 and higher than 0.5 [69]. To confirm the presence of discriminant validity, it was used the confidence interval test [70], which consists of constructing the confidence intervals resulting from the correlations between the different latent variables that compose the CFA model (see **Table 4**). This test indicates the model's discriminant validity, since no

With all of these antecedents, it concluded that the proposes model shows a good degree of

ues established for these reliability parameters are fulfilled in all cases (see **Table 3**).

confidence interval contained the value of 1 [71].

**Table 2.** Exploratory factorial analysis of the scales.

general validity.

102 Marketing

**Table 4.** Discriminant validity.


**12. Breastfeeding**

According to the expert group, breastfeeding is to be avoided because of the potential effects of prolactin subfragments, but in a study where PPCM patients chose to breastfeed, none had adverse maternal effects, and that rate of recovery of left ventricular function was significantly higher in lactating women, and accordingly given the benefits of breastfeeding, it is recommended that women who are stable should continue breastfeeding as long as it is compatible with their heart failure medications [32].


*Edited by Jesus Hamilton Ortiz and Alvaro Pachon de la Cruz*

A mobile ad hoc network (MANET) is a collection of two or more wireless devices with the capability to communicate with each other without the aid of any centralized administrator. Ad hoc networks have no fixed routers, these nodes can be connected dynamically in an arbitrary manner. MANETs, due to their operational characteristics, the dynamics of their changes and the precariousness of their resources, offer huge challenges due to the architecture and service nature in the next generation of mobile communications. MANETs play an important role in the future of next-generation networks. This special collection identifies and studies the most important concerns in MANETs, and includes contributions from researchers, academics, etc.

ISBN 978-953-51-3109-0

Ad Hoc Networks

Photo by StationaryTraveller / iStock


**Meet the editor**

Professor Dr. Krassimir Metodiev, MD, PhD, DScmed, FUICC, FHMO, FNRH, is the Head, Dept. Preclinical and Clinical Sciences, Medical University, Varna-Bulgaria, with specialties: immunology, microbiology, chemotherapy. He has qualified in Germany, Austria, Slovakia, Italy, Holland, Hungary, USA and has over 220 publications, personal impact factor over 165.00, 6 medical

books. Has served as a guest-professor in: LMU, Munich-Germany; Danube University, Krems-Austria; Radium Hospital/Inst. Cancer Research, Oslo-Norway; Hadassah-Jerusalem, Sheba-Tel Aviv and Rambam-Haifa, Israel. Has been invited speaker and chaired symposia and conferences in over 40 countries. Editor-in-chief of Journal of IMAB; Editor and member of Editorial Board: Intl. J. Immunopathology and Pharmacology, Europ. J. Inflammation, CEJM, J. Antimicrobial Agents, J.ISAO. Referent: UICC (oncoimmunology), NATO Scientific Affairs' Division (bioterrorism). President of IMAB, Secretary General of FESCI, Board member of ISAO, ESAO, EANO, ISAO/IFAO, EUCAST, BS-BIG-CIC. Has honorary positions/titles: Honorary Professor and Doctor of Hadassah-Jerusalem, Radium-Oslo, Chiang Mai-Thailand; Honorary Consul of Israel in Bulgaria.

Contents

**Preface IX**

Chapter 2 **"Wrapped Up" Vaccines in**

Chapter 3 **Is Anticancer Vaccine Possible:**

Yıldız Camcıoğlu

Cleo G. Anastassopoulou

Luis Enrique Jerez Puebla

Chapter 7 **Pattern of Clinical Presentations**

Chapter 8 **Selective Antibody Deficiency**

Umezurike Hughes Okafor

Chapter 5 **Chemokine Receptors**

**Section 1 Immunodeficiency – Immunotherapy 1**

Winni De Haes, Charlotte Pollard, Guido Vanham and Joanna Rejman

**CD34+ Blood Progenitor Cells 77**

Mary A. Slatter and Andrew R. Gennery

**the Context of HIV-1 Immunotherapy 27**

**Experimental Application of Produced mRNA Transfected Dendritic Cells Derived from Enriched**

Paula Lazarova, Gunnar Kvalheim and Krassimir Metodiev

Chapter 4 **Immunoglobulin Treatment of Immunodeficient Patients 89**

**Section 2 Immunodeficiency – Mechanisms and Pathophysiology 113**

**as Therapeutic Targets in HIV Infection 115**

Chapter 6 **Fungal Infections in Immunosuppressed Patients 149**

**in Immunocompromised Patient 177**

**with Normal Immunoglobulins 191**

Ricardo U. Sorensen, Tammy Harvey and Lily E. Leiva

Chapter 1 **Stem Cell Transplantation for Primary Immunodeficiency 3**


**Author details**

Ahmed Benchettouh1 \*, Sihem Jebari2 and Lakhdar Kouri3

1 Laboratory Biodiversity and Conservation of Water and the Soils, University Abdelhamid Ibn Badis, Amar Telidji University, Mostaganem, Laghouat, Algeria

2 National Research Institute for Rural Engineering, Water, and Forestry, Ariana, Tunis, Tunisia

3 Laboratory Biodiversity and Conservation of Water and the Soils, University Abdelhamid Ibn Badis, Kharouba, Mostaganem, Algeria

\*Address all correspondence to<EMAIL_ADDRESS>and<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


(**b**)

**Figure 7.** (**a**) Responses to abiotic stresses through yeast dilution bioassay with the wild type strain, S1fa transformed with pRS-416-GFP. Yeast wild type (EV) strain and S1fa overexpressing cells were grown in the URA liquid medium for 24 h at 28 ◦C. The cell solutions were diluted to an OD600 value of 0.3 and exposed to different types of abiotic stresses. (**A**) URA (control), (**B**) 50 mM Cu, (**C**) 75 mM Hg, (**E**) 100 mM Al, (**D**) 100 mM Co, (**F**) 75 μM Cd, (**G**) 2 M mannitol, (**H**) 1 M NaCl. Triangles represent the 10-fold serial dilutions (the starting OD600 is 0.3). (**b**) Responses to temperature stress tolerance through yeast dilution bioassay with the wild type strain, S1fa transformed with pRS-416- GFP. The yeast cells were exposed to (**A**) low temperature (4 ◦C) and (**B**) high temperature (38 ◦C) for 24 h, and then transferred to a place with the normal temperature (28 ◦C). Triangles represent the 10-fold serial dilutions (the starting OD600 is 0.3).


**2.1.5 Binding affinity and complexation thermodynamics**

Once the complex is released from the endosome, DNA must disassemble from its vector to be accessible to the cell machinery responsible for translating the enclosed information. For the case of polyplexes, the DNA decompaction (disassembly) is a process substantially dependent on i) the DNA-vector binding affinity, ii) the complexation thermodynamics, and iii) the solution conditions (Carlstedt et al., 2010; Prevette et al., 2007). Provided that acidic conditions demonstrated to be optimal for polyplex formation (see previous sections), we evaluated the DNA-chitosan binding affinity and complexation thermodynamics at a solution pH of 5.0. As depicted by isothermal titration calorimetry (ITC), lower valence chitosans demonstrated to have a higher binding affinity for DNA. Main results are described below.

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 395

Table 2 summarizes the enthalpy, entropy, binding constant, and the stoichiometry of the

C(689) 29.9 + 0.75 0.27 + 0.03 -2.176 + 0.23 0.022 C(1652) 29.0 + 1.23 0.27 + 0.05 -2.712 + 0.12 0.020 C(2901) 4.82 + 0.08 0.73 + 0.01 -0.598 + 0.07 0.024

C(689) 0.095 + 0.01 0.86 + 0.02 -1.304 + 0.16 0.014 C(1652) 0.011 + 0.02 0.75 + 0.04 -1.357 + 0.12 0.014 C(2901) 0.009 + 0.01 0.62 + 0.07 -0.529 + 0.02 0.012

Reproduced from (Alatorre-Meda et al., 2011) with permission of Elsevier BV in the format Journal via

Table 2. Thermodynamic parameters of the DNA-chitosan binding process. Sub-indices next

As can be observed from this table, the DNA binding constants obtained for all chitosans are on the order of 105 to 106 and 103 to 104 M−1 for the first and second class of binding sites, respectively. These results are in good agreement with previously reported values for other systems including cationic polymers (Nisha et al., 2004; Prevette, et al., 2007) and proteins (Engler et al., 1997; Milev et al., 2005). On the other hand, the decreasing values of the binding constants with the chitosan valence reveal that lower valence chitosans have a higher binding affinity for DNA. This is indicative that chitosan chains may undergo steric restrictions as Mw (valence) increases, restrictions that in turn apparently hamper the interpolyelectrolyte interactions (Danielsen et al., 2004; Maurstad et al., 2007). Concerning the enthalpy, it is well known that it results from a combination of electrostatics, conformational changes (especially for second binding sites), and hydrogen bonding interactions; therefore, ΔH cannot be strictly related to any one contribution. However, and despite the experimental evidence demonstrating that the binding enthalpy ΔH was negative, the DNA–chitosan complexation was proved to be entropically driven. This result is in good agreement with other electrostatic, polyelectrolyte associations promoted by the release of counterions and solvent upon attraction (Matulis et al., 2000; Prevette, et al., 2007;

The potential of chitosans C(689) and C(2901) as DNA carriers towards HeLa cells was evaluated. Polyplexes and nanospheres with compositions in the range 1 ≤ N/P ≤ 18 were tested. In addition, a MEP:DNA lipoplex (4:1 μL:μg) and naked DNA were measured as positive and negative controls, respectively. Although considerably lower compared to that of the DNA–MEP lipoplex, the transfection efficiency of the polyplexes was found to increase with chitosan valence as depicted by the β-galactosidase and luciferase expression

Δ**H1 (kcal mol-1)**

Δ**H2 (kcal mol-1)**

Δ**S1 (kcal mol-1 K-1)**

Δ**S2 (kcal mol-1 K-1)**

DNA–chitosan interaction derived from the data fitting of figure 6.

**(M-1) n1**

**(M-1) n2**

to each parameter stand for the corresponding sites 1 and 2.

**chitosan K1** × **10-5**

**chitosan K2** × **10-5**

Copyright Clearance Center.

Srinivasachari et al., 2007).

**2.1.6 Transfection efficiency**

assays. Main findings are described below.

Reproduced from (Alatorre-Meda, et al., 2011) with permission of Elsevier BV in the format Journal via Copyright Clearance Center.

Fig. 6. Integrated heat of interaction of the titration of chitosan to DNA vs. N/P. Chitosans C(689) (squares), C(1652) (circles), and C(2901) (triangles) were titrated. Solid lines represent the two site model fitting to the experimental data.

Figure 6 shows the heat of interaction resulting from the titration of chitosan to DNA as a function of N/P. Supported on calorimetric measurements, it is well accepted that polyelectrolyte complex formation and coacervation are mainly entropically driven through the release of condensed counterions via the ion-exchange process in which an endothermic signal is recorded during the complex formation (de Kruif et al., 2004; Matulis et al., 2000). By contrast, in a result most commonly observed in the formation of protein–ligand complexes, it can be observed from figure 6 that for all experiments the injection of chitosan appears as a markedly negative signal at the beginning of the binding process followed by a gradual decrease in the released heat up until thermal equilibrium, that is, the complexation is exothermic. A similar decrease in the quantity of heat released on successive injections of titrant has been interpreted as an indicative of the progressive neutralization of charges in the reservoir molecule; meanwhile, the zone of the thermogram in which a plateau in the heat released is reached might be attributed to the complete DNA compaction (Bharadwaj et al., 2006). In this context, comparing the onset of the plateau yielded for the three chitosans during the DNA compaction it is clear that for C(2901) N/P∼1, whereas for C(689) and C(1652) N/P∼0.5. This result is consistent with those we observed by SLS (section 2.1.1) and ζ-potential (previous section) indicating that the use of higher valence chitosans, even at low pHs, apparently demands larger amounts of cationic polymer for DNA compaction. This is also supported by the 4–5-fold smaller enthalpic contribution rendered by C(2901) compared to its lower valence homologues (discussed below).

Table 2 summarizes the enthalpy, entropy, binding constant, and the stoichiometry of the DNA–chitosan interaction derived from the data fitting of figure 6.

Reproduced from (Alatorre-Meda et al., 2011) with permission of Elsevier BV in the format Journal via Copyright Clearance Center.

Table 2. Thermodynamic parameters of the DNA-chitosan binding process. Sub-indices next to each parameter stand for the corresponding sites 1 and 2.

As can be observed from this table, the DNA binding constants obtained for all chitosans are on the order of 105 to 106 and 103 to 104 M−1 for the first and second class of binding sites, respectively. These results are in good agreement with previously reported values for other systems including cationic polymers (Nisha et al., 2004; Prevette, et al., 2007) and proteins (Engler et al., 1997; Milev et al., 2005). On the other hand, the decreasing values of the binding constants with the chitosan valence reveal that lower valence chitosans have a higher binding affinity for DNA. This is indicative that chitosan chains may undergo steric restrictions as Mw (valence) increases, restrictions that in turn apparently hamper the interpolyelectrolyte interactions (Danielsen et al., 2004; Maurstad et al., 2007). Concerning the enthalpy, it is well known that it results from a combination of electrostatics, conformational changes (especially for second binding sites), and hydrogen bonding interactions; therefore, ΔH cannot be strictly related to any one contribution. However, and despite the experimental evidence demonstrating that the binding enthalpy ΔH was negative, the DNA–chitosan complexation was proved to be entropically driven. This result is in good agreement with other electrostatic, polyelectrolyte associations promoted by the release of counterions and solvent upon attraction (Matulis et al., 2000; Prevette, et al., 2007; Srinivasachari et al., 2007).

#### **2.1.6 Transfection efficiency**

394 Non-Viral Gene Therapy

**0.0 0.5 1.0 1.5 2.0 2.5 3.0**

*N/P*

Reproduced from (Alatorre-Meda, et al., 2011) with permission of Elsevier BV in the format Journal via

Fig. 6. Integrated heat of interaction of the titration of chitosan to DNA vs. N/P. Chitosans C(689) (squares), C(1652) (circles), and C(2901) (triangles) were titrated. Solid lines represent

Figure 6 shows the heat of interaction resulting from the titration of chitosan to DNA as a function of N/P. Supported on calorimetric measurements, it is well accepted that polyelectrolyte complex formation and coacervation are mainly entropically driven through the release of condensed counterions via the ion-exchange process in which an endothermic signal is recorded during the complex formation (de Kruif et al., 2004; Matulis et al., 2000). By contrast, in a result most commonly observed in the formation of protein–ligand complexes, it can be observed from figure 6 that for all experiments the injection of chitosan appears as a markedly negative signal at the beginning of the binding process followed by a gradual decrease in the released heat up until thermal equilibrium, that is, the complexation is exothermic. A similar decrease in the quantity of heat released on successive injections of titrant has been interpreted as an indicative of the progressive neutralization of charges in the reservoir molecule; meanwhile, the zone of the thermogram in which a plateau in the heat released is reached might be attributed to the complete DNA compaction (Bharadwaj et al., 2006). In this context, comparing the onset of the plateau yielded for the three chitosans during the DNA compaction it is clear that for C(2901) N/P∼1, whereas for C(689) and C(1652) N/P∼0.5. This result is consistent with those we observed by SLS (section 2.1.1) and ζ-potential (previous section) indicating that the use of higher valence chitosans, even at low pHs, apparently demands larger amounts of cationic polymer for DNA compaction. This is also supported by the 4–5-fold smaller enthalpic contribution rendered by C(2901) compared to its lower valence

**-3000**

homologues (discussed below).

the two site model fitting to the experimental data.

Copyright Clearance Center.

**-2500**

**-2000**

**-1500**

*Q***, cal mol-1**

**-1000**

**-500**

**0**

The potential of chitosans C(689) and C(2901) as DNA carriers towards HeLa cells was evaluated. Polyplexes and nanospheres with compositions in the range 1 ≤ N/P ≤ 18 were tested. In addition, a MEP:DNA lipoplex (4:1 μL:μg) and naked DNA were measured as positive and negative controls, respectively. Although considerably lower compared to that of the DNA–MEP lipoplex, the transfection efficiency of the polyplexes was found to increase with chitosan valence as depicted by the β-galactosidase and luciferase expression assays. Main findings are described below.

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 397

The luciferase assay was conducted in order to confirm the results obtained by the βgalactosidase assay. The transfection efficiency for all cases was similar to that observed in the β-galactosidase method. In general, naked DNA and complexes regardless of the N/P ratio and structure yielded a luminescence three orders of magnitude lower than that of the DNA–MEP lipoplex. Concerning the protein content determined by the BCA assay, the polyplexes and nanospheres rendered protein contents slightly higher than that of the DNA–MEP lipoplex (data not shown). In consequence, the transfection efficiency of

In general, the low capacity of DNA to escape from complexes is regarded as one of the major limitations for the transfection efficiency of polyplexes (Midoux et al., 2009; Tros de Ilarduya et al., 2010). This feature has been ascribed to a number of factors. While some authors support that an excess of polycation, even in the presence of chloroquine, limits the protein expression due to in vitro cytotoxicity (Fischer et al., 2004), other authors affirm that the low tolerance of DNA to dissociate from polyplexes is presumably due to the bulky form they adopt in solution (Izumrudov et al., 1999). In our case the low transfection efficiency of the DNA–chitosan polyplexes compared to that of the lipoplex is most likely related to both the colloidal properties they exhibit and the DNA-chitosan binding affinity. On the one hand, the morphological structure depicted by TEM and AFM in conjunction with the markedly positive ζ-potentials obtained suggest a core–shell like polyplex structure with chitosan occupying the outer part of the complex (A. V. Kabanov & V. A. Kabanov, 1998). On the other hand, as mentioned before, chitosans presenting higher DNA binding affinities were found to yield lower transfection

In general, the DNA-chitosan polyplexes exhibited good colloidal properties such as sizes in the range of 180 to 250 nm, ζ-potentials of about 16 mV, and a stable core-shell like structural conformation. The influence of chitosan charge density and valence on all these

Chitosan charge density was found to play an important role on the complexation of DNA. Namely, as the solution pH got close to the chitosan pKa, the neutralization of the amino groups, entailing a decrease of chitosan charge density, resulted in a higher amount of chitosan needed for complexation. That is, the acidic conditions were found to be favorable

Chitosan valence was found to be related to both the polyplex size and, more importantly, to the transfection efficiency. On the one hand, the hydrodynamic radii of complexes increased linearly with the chitosan Mw demonstrating that larger chitosan chains produce bulkier and less soluble polyplexes. On the other hand, in what we conceive as the main contribution of our investigations, we found that higher valence chitosans, exhibiting lower

As stated before, cationic polymers most commonly studied as gene carriers include chitosan, PEI, PLL, poly(β-amino ester)s, and poly-(amidoamine) dendrimers. In addition,

polyplexes was confirmed to be low compared to that of the lipoplex.

efficiencies.

**2.1.7 Particular conclusions**

for complex formation. 2. Role of chitosan valence.

1. Role of chitosan charge density.

**2.2 The DNA-pDADMAC system**

physicochemical properties can be summarized as follows.

DNA binding affinities, yielded higher DNA transfection efficiencies.

Figure 7 shows the transfection efficiency of the polyplexes and controls as well as the protein content of the wells after lysation. Two features are observed from this figure. On the one hand, it is clear that polyplexes within the whole range of ratios rendered levels of βgalactosidase expression slightly higher than that of the negative control (DNA without polymer) with transfection efficiency being increased with chitosan valence. This result is somehow logical taking into account the lower binding affinity depicted by ITC assays for the DNA–C(2901) complex (see Table 2); that is, the DNA release from this complex in the interior of the cell is expected to be favored. On the other hand, compared to that of the DNA–MEP lipoplex, the transfection efficiency of the polyplexes is considerably lower.

Reproduced from (Alatorre-Meda et al., 2011) with permission of Elsevier BV in the format Journal via Copyright Clearance Center.

Fig. 7. Transfection efficiency of the DNA-chitosan complexes (columns) and protein content in wells after lysation (squares) vs. N/P.

Speculating that the low transfer rate of the polyplexes (compared to that of the lipoplex) might be related to cytotoxicity effects, the protein concentration was determined via the BCA assay. From figure 7 (right hand side axis) a comparable level of protein content for all formulations including the blanks was observed. Even if not totally conclusive regarding a measure of the cytotoxicity, this result shows that the cells proliferated approximately in the same way; consequently, the poor transfection efficiency shown by the DNA–chitosan polyplexes cannot be ascribed to cytotoxicity.

In the light of the transfection efficiency depicted by the polyplexes, a second kind of experiment was implemented. DNA–chitosan nanospheres were prepared following the protocol described by Mao and coworkers (Mao et al., 2001). The transfection efficiency results for C(689) are also plotted in figure 7. Compared to the polyplexes, no transfection improvement was observed, on the contrary, nanospheres in general showed a decreasing in gene expression yielding results only comparable to the naked DNA administration.

The luciferase assay was conducted in order to confirm the results obtained by the βgalactosidase assay. The transfection efficiency for all cases was similar to that observed in the β-galactosidase method. In general, naked DNA and complexes regardless of the N/P ratio and structure yielded a luminescence three orders of magnitude lower than that of the DNA–MEP lipoplex. Concerning the protein content determined by the BCA assay, the polyplexes and nanospheres rendered protein contents slightly higher than that of the DNA–MEP lipoplex (data not shown). In consequence, the transfection efficiency of polyplexes was confirmed to be low compared to that of the lipoplex.

In general, the low capacity of DNA to escape from complexes is regarded as one of the major limitations for the transfection efficiency of polyplexes (Midoux et al., 2009; Tros de Ilarduya et al., 2010). This feature has been ascribed to a number of factors. While some authors support that an excess of polycation, even in the presence of chloroquine, limits the protein expression due to in vitro cytotoxicity (Fischer et al., 2004), other authors affirm that the low tolerance of DNA to dissociate from polyplexes is presumably due to the bulky form they adopt in solution (Izumrudov et al., 1999). In our case the low transfection efficiency of the DNA–chitosan polyplexes compared to that of the lipoplex is most likely related to both the colloidal properties they exhibit and the DNA-chitosan binding affinity. On the one hand, the morphological structure depicted by TEM and AFM in conjunction with the markedly positive ζ-potentials obtained suggest a core–shell like polyplex structure with chitosan occupying the outer part of the complex (A. V. Kabanov & V. A. Kabanov, 1998). On the other hand, as mentioned before, chitosans presenting higher DNA binding affinities were found to yield lower transfection efficiencies.

### **2.1.7 Particular conclusions**

396 Non-Viral Gene Therapy

Figure 7 shows the transfection efficiency of the polyplexes and controls as well as the protein content of the wells after lysation. Two features are observed from this figure. On the one hand, it is clear that polyplexes within the whole range of ratios rendered levels of βgalactosidase expression slightly higher than that of the negative control (DNA without polymer) with transfection efficiency being increased with chitosan valence. This result is somehow logical taking into account the lower binding affinity depicted by ITC assays for the DNA–C(2901) complex (see Table 2); that is, the DNA release from this complex in the interior of the cell is expected to be favored. On the other hand, compared to that of the DNA–MEP lipoplex, the transfection efficiency of the polyplexes is considerably lower.

**MEP 4:1**

in wells after lysation (squares) vs. N/P.

polyplexes cannot be ascribed to cytotoxicity.

**0.00**

Copyright Clearance Center.

**0.01**

**0.02**

**0.03**

**Transfection efficiency, mg-1 s-1**

**0.04**

**0.05**

**0.06**

**--**

**N/P 1**

**N/P 2**

**N/P 3**

**N/P 6**

**N/P 12**

**N/P 18**

**--**

**N/P 1**

Reproduced from (Alatorre-Meda et al., 2011) with permission of Elsevier BV in the format Journal via

Fig. 7. Transfection efficiency of the DNA-chitosan complexes (columns) and protein content

Speculating that the low transfer rate of the polyplexes (compared to that of the lipoplex) might be related to cytotoxicity effects, the protein concentration was determined via the BCA assay. From figure 7 (right hand side axis) a comparable level of protein content for all formulations including the blanks was observed. Even if not totally conclusive regarding a measure of the cytotoxicity, this result shows that the cells proliferated approximately in the same way; consequently, the poor transfection efficiency shown by the DNA–chitosan

In the light of the transfection efficiency depicted by the polyplexes, a second kind of experiment was implemented. DNA–chitosan nanospheres were prepared following the protocol described by Mao and coworkers (Mao et al., 2001). The transfection efficiency results for C(689) are also plotted in figure 7. Compared to the polyplexes, no transfection improvement was observed, on the contrary, nanospheres in general showed a decreasing in gene expression yielding results only comparable to the naked DNA administration.

**N/P 2**

**N/P 3**

**nanospheres DNA-C(689) DNA-C(2901)**

**N/P 6**

**N/P 12**

**N/P 18**

**--**

**N/P 1**

**N/P 6**

DNA-**C(689)**

**N/P 18**

**--**

**DNA**

**0**

**25**

**50**

 **Protein content per well,**

μ**g**

**75**

**100**

**125**

In general, the DNA-chitosan polyplexes exhibited good colloidal properties such as sizes in the range of 180 to 250 nm, ζ-potentials of about 16 mV, and a stable core-shell like structural conformation. The influence of chitosan charge density and valence on all these physicochemical properties can be summarized as follows.

1. Role of chitosan charge density.

Chitosan charge density was found to play an important role on the complexation of DNA. Namely, as the solution pH got close to the chitosan pKa, the neutralization of the amino groups, entailing a decrease of chitosan charge density, resulted in a higher amount of chitosan needed for complexation. That is, the acidic conditions were found to be favorable for complex formation.

2. Role of chitosan valence.

Chitosan valence was found to be related to both the polyplex size and, more importantly, to the transfection efficiency. On the one hand, the hydrodynamic radii of complexes increased linearly with the chitosan Mw demonstrating that larger chitosan chains produce bulkier and less soluble polyplexes. On the other hand, in what we conceive as the main contribution of our investigations, we found that higher valence chitosans, exhibiting lower DNA binding affinities, yielded higher DNA transfection efficiencies.

#### **2.2 The DNA-pDADMAC system**

As stated before, cationic polymers most commonly studied as gene carriers include chitosan, PEI, PLL, poly(β-amino ester)s, and poly-(amidoamine) dendrimers. In addition,

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 399

in the conductivity plot for the DNA one (plots not shown). The ratio at which the conductivity inflection occurred is reported as (N/P)c (see section 2.1.1 for a complete

It is clear from table 4 that (N/P)c is governed by the charge density provided that polyplexes formed with pDADMAC homopolymers have complexation ratios lower than that of the polyplex formed with coDADMAC. This result confirms what we observed for chitosan not only with respect to the role of charge density but also regarding the (N/P)c values obtained which are very similar for both chitosan- and pDADMAC-based polyplexes

**polymer (N/P)c (N/P)\* RH (nm)**

**p(1, < 619)** 0.7 4 79.4 + 2.1

**p(1, 929)** 0.6 2 87.0 + 6.9

**p(1, 1703)** 0.7 2 108.9 + 12.5

**p(1, 2786)** 0.6 1 112.6 + 9.7

**p(0.26, 668)** 1.5 2 199.8 + 23.5

Reproduced from (Alatorre-Meda et al., 2010b) with permission of AMERICAN CHEMICAL SOCIETY

Table 4. Characteristic N/P ratios and RH of the DNA-pDADMAC polyplexes. (N/P)c and (N/P)\* stand for the DNA compaction ratio (determined by conductometry) and for the ratio from which the size of the polyplexes remain constant (determined by DLS), respectively. RH is the average of the recorded values in the range (N/P)\* ≤ N/P ≤ 10.

(N/P)\* was found by DLS as a characteristic ratio from which the polyplexes adopt the most compact structure. As observed from table 4, the value of (N/P)\* follows a decreasing trend with pDADMAC valence of the polyplexes, with p(1,619) and p(1,2786) showing the highest and the lowest (N/P)\* values of 4 and 1, respectively. The interplay between (N/P)c and (N/P)\* can be reasoned in terms of the different complexation states of DNA mediated by

The characterization of the DNA-pDADMAC polyplexes in terms of size and time stability was carried out by means of DLS. The study was done in two steps. First, the hydrodynamic radii of the polyplexes, RH, were determined at 0.2 ≤ N/P ≤ 10. And second, the evolution of RH with time was followed for the polyplexes at N/P = 10. RH results are depicted in table 4. It is observed from table 4, that similarly to the chitosan systems, the size of the polyplexes was found to increase with pDADMAC valence, that is, the general assumption that the electrostatic interactions are outweighed to a certain extent by a decrease in the polycation solubility is confirmed (MacLaughlin et al., 1998; Mumper et al., 1995). Concerning the coDADMAC polyplex, it is clear that its size is ca. twice as big as those of the pDADMAC polyplexes. This outcome can be a consequence of an expected lower degree of DNA

in the format Journal via Copyright Clearance Center.

pDADMAC (Fischer et al., 2004).

**2.2.2 Time stability and size**

explanation).

(see section 2.1.1).

because of its permanent cationic charge, poly(diallyldimethylammonium chloride) (pDADMAC) has recently been explored as well (Fischer et al., 2004; Krajcik et al., 2008). pDADMAC is a water soluble cationic polymer. It is composed of mainly configurational isomers of pyrrolidinium rings and a small amount of pendant double bonds (Dautzenberg et al., 1998; Jaeger et al., 1996). With the pendent allylic double bonds being less reactive than those of the monomer, strictly linear macromolecules are formed at low conversions, but branching can proceed at high conversions as was demonstrated for commercial samples (Wandrey et al., 1999). Because of its physical structure pDADMAC is a highly flexible polymer compared to other polycations such as chitosan (Marcelo et al., 2005; Trzcinski et al., 2002). pDADMAC has been widely used in technical applications as a flocculant agent and as a composite for biosensors, which is because of its pH-independent cationic charge (Dautzenberg et al., 1998; Jaeger et al., 1996).

Table 3. pDADMACs employed. In p(x,y), x and y stand for charge density and valence, respectively.

In the present section we summarize outstanding results obtained in our laboratory describing relevant physicochemical characteristics of the DNA-pDADMAC complexes (Alatorre-Meda et al., 2010b). As done for chitosan in the previous section, we highlight the role of pDADMAC charge density and valence. Four homo-polymers (charge density = 1, with different valences) and one co-polymer, p(acrylamide-co-diallyldimethylammonium chloride) (coDADMAC) (charge density < 1, equivalent in valence to one of the homopolymers), were employed. Table 3 lists the cationic polymers characterized as gene carriers along with the nomenclature cited throughout this section.


**2. Resistance evolution background**

*Staphylococcus* spp. are gram-positive cocci, catalase positive, belonging to the family Staphylococcaceae and the genus *Staphylococcus,* which currently comprises 45 species (Euzéby, 2011), from which 17 species can be isolated from samples from humans. *Staphylococcus aureus* is the most important species and can be found in both healthy and immunocompromised individuals (Santos et al., 2007).

The *Staphylococcus* genus is classified according to the synthesis of the enzyme coagulase, and those that synthesize are classified as coagulase-positive, represented by the species *Staphylococcus aureus, S. intermedius, S. hyicus, S. schleiferi* subspecie *coagulans*, *S. delphine* and *S. lutrae,* and in the absence of the synthesis, are represented by other coagulase-negative species (Baba et al., 2002). Both groups can cause infections in humans and, among them, the main species are: *S. aureus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. hominis, S. warneri, S. capitis, S. saccarolyticus, S. lugdunensis, S. cohnii, S. xylosus, S. simulans, S. auricularis, S. caprae* and *S. schleiferi* (Layer et al., 2006).

Humans are the main reservoir of *Staphylococcus aureus,* which can colonize the skin, throat, intestine and nasal passages without causing damage to the host. Asymptomatic carriers in hospitals can spread *S. aureus* increasing risk to immunocompromised patients (Santos et al., 2007). This ubiquity favors the installation of various types of infections such as necrotizing pneumonia, skin and soft tissue infections, food poisoning bacteremia through the synthesis of enterotoxin (Santos et al., 2007; Cunha et al., 2006; Jarraud et al., 2002; Gandhinagar & Silva, 2004).

In 1940, staphylococcal infections were treated with penicillin; however, only two years after its introduction, nosocomial penicillinase producing strains grew resistant to penicillin by the inactivation of the penicillin molecules (Mimica & Mendes, 2007). Shortly thereafter, the same occurred with the strains of community origin necessitating the use of alternative antibiotics to treat infections caused by *S. aureus* (Ricardo, 2004).

In the late 1950s, in Europe, resistant nosocomial and community *Staphylococcus* spp. had penicillin resistance rates of 90% and 70%, respectively (Ricardo, 2004). This finding led to a

can be classified in a range from type I to XI largely used to discriminate hospital and

Community strains are usually more sensitive to other antimicrobial classes compared to multiresistant health care associated strains. In fact, CA-MRSA can be resistant to other antimicrobials besides beta-lactams, thus decreasing the options of drug choices. Another subject of concern is resistance to the drug of choice for treating MRSA, vancomycin. There are few antimicrobials to control the infections caused by MRSA and vancomycin is one of them. Reports of resistance and low level of sensitivity to this drug in health care

All of the issues mentioned above, underscore the importance of further research on the behavior of CA-MRSA at the epidemiological and molecular levels enabling establishment and application of preventive measures and treatment in view of infections. The present chapter will outline the main features of CA-MRSA and epidemiological principles involved

*Staphylococcus* spp. are gram-positive cocci, catalase positive, belonging to the family Staphylococcaceae and the genus *Staphylococcus,* which currently comprises 45 species (Euzéby, 2011), from which 17 species can be isolated from samples from humans. *Staphylococcus aureus* is the most important species and can be found in both healthy and

The *Staphylococcus* genus is classified according to the synthesis of the enzyme coagulase, and those that synthesize are classified as coagulase-positive, represented by the species *Staphylococcus aureus, S. intermedius, S. hyicus, S. schleiferi* subspecie *coagulans*, *S. delphine* and *S. lutrae,* and in the absence of the synthesis, are represented by other coagulase-negative species (Baba et al., 2002). Both groups can cause infections in humans and, among them, the main species are: *S. aureus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. hominis, S. warneri, S. capitis, S. saccarolyticus, S. lugdunensis, S. cohnii, S. xylosus, S. simulans, S.*

Humans are the main reservoir of *Staphylococcus aureus,* which can colonize the skin, throat, intestine and nasal passages without causing damage to the host. Asymptomatic carriers in hospitals can spread *S. aureus* increasing risk to immunocompromised patients (Santos et al., 2007). This ubiquity favors the installation of various types of infections such as necrotizing pneumonia, skin and soft tissue infections, food poisoning bacteremia through the synthesis of enterotoxin (Santos et al., 2007; Cunha et al., 2006; Jarraud et al., 2002; Gandhinagar &

In 1940, staphylococcal infections were treated with penicillin; however, only two years after its introduction, nosocomial penicillinase producing strains grew resistant to penicillin by the inactivation of the penicillin molecules (Mimica & Mendes, 2007). Shortly thereafter, the same occurred with the strains of community origin necessitating the use of alternative

In the late 1950s, in Europe, resistant nosocomial and community *Staphylococcus* spp. had penicillin resistance rates of 90% and 70%, respectively (Ricardo, 2004). This finding led to a

community types.

Silva, 2004).

environments have been reported worldwide.

in the outcome briefly described above.

**2. Resistance evolution background**

immunocompromised individuals (Santos et al., 2007).

*auricularis, S. caprae* and *S. schleiferi* (Layer et al., 2006).

antibiotics to treat infections caused by *S. aureus* (Ricardo, 2004).

search for alternatives, and in 1959, adding the acid 6-aminopenicilanic in the penicillin molecule allowed for protecting the precursor of penicillin beta-lactam ring. This semisynthetic penicillin (Named methicillin (and the analog oxacillin, used in Brazil) proved to be resistant to the action of beta-lactamase. However, both were effective for a short period of time, and in 1961, strains resistant to semisynthetic penicillins emerged. These emerging new strains were named MRSA (Methicillin-Resistant *Staphylococcus aureus*), so far unique to hospital settings (Ricardo, 2004; Salgado et al., 2003).

In the 1980s the first reports emerged of infections caused by *S. aureus* in patients without risk factors for acquisition of nosocomial MRSA (HA-MRSA), resulting in the designation of CA-MRSA (Community-Acquired Methicillin-Resistant *S. aureus*) in the 1990s when reports of CA-MRSA increased (Ricardo, 2004). The community-acquired infections are usually distinct from hospitals in terms of susceptibility and the carriage of the gene that codifies for the synthesis of Panton-Valentine Leukocidin (PVL) responsible for tissue invasion preceding the skin infections (Klevens et al., 2007).

According to criteria, it is considered that individuals affected by CA-MRSA should not report previous MRSA infections; the patient must have a positive culture for MRSA within 48 hours after hospital admission, and must not be hospitalized in the last 12 months, or admitted to nursing homes or homecare, and did not report undergoing dialysis, surgery, catheters or any prior or invasive treatment at the time of MRSA isolation (Salgado et al. 2003).

The transmission of the bacteria occurs through direct contact of susceptible individuals with asymptomatic carriers.

The transmission of MRSA among family members was reported in a study with 10 families. Strains with PFGE ST8 (USA 300), ST59 (USA 1000), and ST80 PVL-positive were found in this investigation (Huijsdens et al., 2006). Lung infections caused by these strains can be serious because the symptoms are similar to pneumonia in children; therefore, it is important to consider infection by MRSA especially if there were previous reported. (Huijsdens et al., 2006).

Among pathogens that can cause pulmonary infections, CA-MRSA is associated with pneumonia and is frequently associated with pulmonary viral coinfection. Studies indicate that CA-MRSA agents are commonly found in pneumonia during the influenza season, and about 85% of *S. aureus* strains isolated contained the PVL gene. In three cases of severe necrotizing pneumonia, strategies were carried out to prevent the synthesis of Leukocidin, including the administration of antibiotics such as rifampicin, linezolid and clindamycin, and/or applying intravenous immunoglobulin to block the lytic effect of PVL under polymorphonuclear cells (Rouzic et al., 2010). During the influenza season of 2006 and 2007, there were 51 reported cases of pneumonia in health centers caused by *S. aureus*, and 79% were MRSA. Most cases of pneumonia due to MRSA occurred during or after viral infection (Kallen et al., 2008).

CA-MRSA: Epidemiology of a Pathogen of a Great Concern 57

Persistent carriers are usually colonized by a single strain of *S. aureus* over a long period of time, whereas intermittent carriers may carry different strains over time (Werthein et al., 2005). MRSA colonization in individuals in the community remains a low burden as demonstrated in a study of high school boys where no individuals were found colonized with MRSA. The fact that there were no carriers among this population may be a reflection of improvement in hygiene practices among these individuals due to previous reports of outbreaks in team sports (Lear et al., 2011). Although they did not found MRSA in the population, other studies have found colonization rates in individuals in the community

MRSA colonization in a hospital environment is a matter of utmost importance since it is characterized as a predisposing factor to infection. Nasal decolonization is usually performed with the application of mupirocin and is useful for reducing symptoms and its spread in hospital environments. However, the practice of decolonization with antimicrobials remains controversial because of the risk of acquiring drug resistance, which limits its use. Despite this risk, it is advised to perform decolonization in healthcare settings because of the risk of developing infections especially in individuals who are under invasive

But how should we manage individuals living in the community who are characterized as persistent carriers of CA-MRSA, but show no clinical manifestations and are healthy? This is a controversial subject because a previous study showed that individuals might be cocolonized with MSSA and MRSA, where the strains of MSSA have better fitness than MRSA, most likely due to the additional mechanism of resistance, which requires a cost in the feasibility and competitiveness of these strains. Thus, when decolonization is performed with mupirocin, both are eliminated and there will be competition, thus increasing the chances of colonization by resistant strains if both were competing for the same ecological niche (Dall'Antonia et al., 2005). Studies are needed to evaluate the cost-effectiveness of nasal colonization among residents in the community as a predisposition to infections, but

The nasal vestibule is composed of highly keratinized cells including apocrine and sebaceous glands and hair follicles. These factors are poorly studied compared to the mucosa and its linkage to mucins. Some of the pathogen virulence factors contribute to successful colonization; for example, the clumping factor B is highly associated with nasal colonization (Wertheim et al., 2007). Studies have reported the binding of *S. aureus* surface protein G (SasG) to a ligand in nasal epithelial cells. Other factors such as: Teichoic acid and cell wall components recognizing microbial surface adhesive matrix molecules (MSCRAMMS) responsible for the adherence protein in fibronectin, fibrinogen and collagen,

Case reports of CA-MRSA emerged worldwide revealing the severity, spread, which has helped to chart the epidemiologic distribution in the various communities and provide a better understanding of the behavior of virulence and resistance profile of these strains involved. Such isolates are associated with diseases of skin and soft tissue (Ribeiro et al., 2005). The infections develop from the skin surface where they penetrate to deeper layers,

ranging from 0.8 to 3% (Keuhnert et al., 2006; Salgado et al., 2003; Ellis et al., 2004).

treatments and are immunocompromised (Coates et al., 2009).

there are chances of acquisition of resistant strains.

**4. Case reports**

may play an important role in colonization (Wertheim et al., 2005).

The treatment of MRSA infections is variable. Within a hospital environment, there are several options such as linezolid, daptomycin, quinupristin/dalfopristin, but vancomycin is one of the most commonly used to treat several types of infections, but the emergence of strains resistant to this antimicrobial agent limits its use. Historically the emergence of vancomycin resistance occurred primarily in isolates of *Enterococcus* spp. in 1986 and reported only in 1988 in a European hospital. In 1989, in the United States, Vancomycin-Resistant Enterococci (VRE) were detected in clinical isolates and in 1993 accounted for 7.9% of the enterococci samples in nosocomial environments reported by the CDC. The most important reservoir is the gastrointestinal tract and transmission occurs mainly through contact with healthcare workers, and indirectly by contaminated hands in contact with the hospital objects where at least one of the patients had diarrhea (Mayall, 2002).

Vancomycin resistance in VRE is due to the presence of *van* genes (A to G) that encode for the synthesis of peptidoglycan by an alternative pathway that produces precursors ending in D-Ala-D-Lac or D-Ala-D-Ser instead of D-Ala-D-Ala. In *S. aureus,* however the main mechanism involved in vancomycin resistance relies on a thickened cell wall, production of abundant extracellular material that remains not well characterized and it ends up compromising the ability of division. These characteristics result in the synthesis of an altered peptidoglycan with an increased number of terminal D-Ala-Ala-D capable of binding free vancomycin in the outer cell wall, thus leading to a lower availability of the antimicrobial target molecule in the intracellular region. Strains of *S. aureus* with intermediate resistance to vancomycin (VISA) may contain 2 to 4 times more layers of D-Ala-D-Ala than susceptible strains, being capable of binding to three to six times more vancomycin molecules. Some chromosomal changes are necessary to maintain this resistance , and in addition require a larger amount of precursors than normal strains, thus compromising their fitness in an environment free of this antimicrobial. This may explain the reason for the loss of the vancomycin resistance of VISA strains when they are in environments without antibiotics, giving rise to heteroresistant strains called hetero-VISA (Van Bambeke et al., 2004).