**Intrinsically Disordered Proteins and Chronic Diseases**

Printed Edition of the Special Issue Published in *Biomolecules* Prakash Kulkarni and Vladimir N. Uversky Edited by

www.mdpi.com/journal/biomolecules


**10. Industrial reaction to produce fertiliser**

Question: According to the following equation, sulphuric acid (H2SO4) reacts with ammonia (NH3) to produce the fertiliser ammonium sulphate ((NH4)2SO4):

(NH4)2SO4 = H2SO4(aq) + 2NH3(g) (aq)

How much ammonium sulphate can be made from 2.0 kg of sulphuric acid and 1.0 kg of ammonia?

Answer

Step 1: Convert the sulphuric acid and ammonia masses into moles n(H2SO4) = m/M

=2000 g/98.078 g/mol = 20.39 mol n(NH3) = 1000 g/17.03 g/mol = 58.72 mol =1000 g/17.03 g/mol = 58.72 mol

Step 2: Determine which of the reactants is limiting using the balanced equation. According to the balanced chemical equation, one mole of H2SO4 reacts with two moles of NH3 to produce one mole of (NH4)2SO4. As a result, 20.39 moles of H2SO4 must react with 40.78 moles of NH3. In this case, NH3 is in excess, and H2SO4 is the limiting factor.

In the following equation, sulphuric acid (H2SO4) reacts with ammonia (NH3) to produce the fertiliser ammonium sulphate ((NH4)2SO4):

H2SO4(aq) + 2NH3(g) = (NH4)2SO4 (aq)

*Implementation of Conceptual Change Approach to Improve Learners' Understanding… DOI: http://dx.doi.org/10.5772/intechopen.114094*

What is the maximum mass of ammonium sulphate that can be obtained from 2.0 kg of sulphuric acid and 1.0 kg of ammonia?

Answer

Step 1: Convert the mass of sulphuric acid and ammonia into moles n.

Step 3: Determine the maximum amount of ammonium sulphate that can be produced.

Answer

According to the equation, the mole ratio of H2SO4 in the reactants to (NH4)2SO4 in the product is 1:1. As a result, 20.39 moles of H2SO4 will produce 20.39 moles of (NH4)2SO4.

The maximum mass of ammonium sulphate that can be produced is calculated as follows:

m = n M = 20.41 mol 132 g/mol = 2694 g

The maximum amount.

The teacher further asked a question referring to example 9,

Why is it necessary to produce fertilisers? [Teacher].

For the production of good quality food. [G3L2].

How the necessary concepts of quantitative aspects of chemical change were clearly explained by the teacher with clarifications that industry productivity also depends on these critical aspects of change.

After the intervention post-test was administered, the post-test was identical to the pre-test. The results show that learners' performance in a post-test improved significantly when compared to the pre-test. The results are analysed and interpreted using a table and graph.

A close examination of the post-test reveals that the intervention process had a positive influence on the results. 60% of the learners achieved more than 50%, indicating that more learners understood the concept. Only 20% got less than 30%.

10% got between 30% and 40%.

### **11. Interviews**

Focus group interviews were conducted with 12 Grade 11 Physical Sciences learners for this study. Three groups of learners were formed. The category of these three formed by four learners in each were coded as (FL). The interviews were conducted to determine how well learners have gained new knowledge through collaborating with others when experiments were conducted. In the group of 50 learners, there was a mix of varying learner abilities from low to high achievers.

At the conclusion of the intervention, all learners took part in focus group interviews. The goal was to get their thoughts on how to implement the lesson. During interviews, learners were asked how they performed in the pre-test versus the posttest, as well as what motivated their responses. According to the responses, all of them scored higher on the post-test because of how the lesson was presented. They compared the lesson presentation to how it is usually taught. The following codes were obtained based on the responses of the learners: activities, experiments, learner participation, group activities, writing activities and approach preference (**Table 4**).

Learners reported that the experiments and demonstrations done in class were the main difference, as they had never done science experiments before. One learner stated that the way the teacher presented the lesson made them grasp all the

#### **Table 4.**

*Learners distribution in percentages indicating codes identified from data gathered.*

information, while the other stated that it was their first time doing experiments in class. All learners stated that they usually made notes, but this time, they wrote laboratory reports, which increased their knowledge retention. In terms of teacherlearner engagements, it was reported by learners that it was their norm to listen and form self-compiled notes, concurrently; for this lesson, there was maximum learner participation. Learners also shared that all learners engaged themselves in discussions as they were eager to see the experiment outcomes. This is supported by one learner who uttered: 'there was exchange of ideas throughout the discussions'.

As indicated by some learners, there were improved collaborations as learners were exposed to working with class members who never belonged to their groups. This has led to the formation of new friends and the extension of social skills. Another achieved skill, as perceived by learners, was compiling reports of what they observed, which was seen to have improved their writing capabilities.

Another learner indicated that they enjoyed writing by themselves. All of them preferred the way the researchers presented the lesson, citing reasons such as learning better, being motivated, enjoying the lesson, participation and involvement, and retention of new knowledge.

### **12. Discussion**

In this investigation, a conceptual change approach was administered to have a better understanding of quantitative aspects versus chemical change.

The study found that there was an improvement in learners' scientific understanding after pre-test and post-test. The study recommends a well-designed conceptual change instructional approach that leads to significantly better acquisition of scientific concepts. The study is significant because it addresses the issue of poor performance in Physical Sciences, particularly in the Eastern Cape Province of South Africa. The study suggests that a conceptual change instructional strategy is an effective teaching strategy that can be used to improve learners' understanding of science concepts. The study also highlights the importance of designing learning environments that allow learners to become aware of their current internal justifications and beliefs, which is essential for promoting conceptual change and improving problem-solving abilities. The study employed a qualitative research methodology, which is appropriate for exploring learners' misconceptions and difficulties on quantitative aspects of chemical change. The study used a case study approach and collected data from 50 learners and 12 teachers. The study used the Chemistry Achievement Test (CAT) to create lessons for the intervention programme and to determine the learners' alternate conceptions. The CAT examinations were piloted on a small sample of Grade 11 Physical Sciences teachers from neighbouring schools to confirm topic validity. The study's findings suggest that the conceptual change approach is an effective teaching

### *Implementation of Conceptual Change Approach to Improve Learners' Understanding… DOI: http://dx.doi.org/10.5772/intechopen.114094*

strategy that can be used to improve learners' understanding of science concepts. The study recommends a well-designed conceptual change instructional approach that leads to significantly better acquisition of scientific concepts. The study also highlights the importance of designing learning environments that allow learners to become aware of their current internal justifications and beliefs, which is essential for promoting conceptual change and improving problem-solving abilities. Overall, this study provides valuable insights into the use of conceptual change instructional strategy to improve learners' understanding of science concepts [14]. The study's findings have important implications for science education in South Africa and other countries facing similar challenges. The study suggests that science educators should consider using conceptual change instructional strategy to improve learners' understanding of science concepts and promote problem-solving abilities.

### **13. Findings**

The conceptual change approach was effective: The study found that the conceptual change approach was an effective teaching strategy that led to a significant improvement in learners' scientific understanding after pre-test and post-test. This means that learners who participated in the conceptual change instructional programme had a better understanding of the quantitative aspects of chemical change than those who did not participate in the programme.

Importance of designing learning environments: The study highlighted the importance of designing learning environments that allow learners to become aware of their current internal justifications and beliefs, which is essential for promoting conceptual change and improving problem-solving abilities. This means that teachers should create a classroom environment that encourages learners to reflect on their existing ideas and beliefs about a topic so that they can challenge and change them when necessary.

Qualitative research methodology: The study employed a qualitative research methodology, which is appropriate for exploring learners' misconceptions and difficulties on quantitative aspects of chemical change. This means that the study used open-ended questions and interviews to collect data on learners' understanding of the topic, rather than relying on standardised tests or surveys.

Case study approach: The study used a case study approach, which involved studying a particular group of learners or a specific classroom setting in depth. This allowed the researchers to get a detailed understanding of the learners' existing ideas and beliefs, as well as the effectiveness of the conceptual change instructional programme.

Use of Chemistry Achievement Test (CAT): The study used the Chemistry Achievement Test (CAT) to create lessons for the intervention programme and to determine the learners' alternate conceptions. The CAT examinations were piloted on a small sample of Grade 11 Physical Sciences teachers from neighbouring schools to confirm topic validity. This means that the study used a standardised test to assess learners' understanding of the topic and to create lessons that addressed their specific misconceptions.

Implications for science education: The study's findings have important implications for science education in South Africa and other countries facing similar challenges. The study suggests that science educators should consider using the conceptual change instructional strategy to improve learners' understanding of science concepts and promote problem-solving abilities. This means that science teachers

should incorporate a conceptual change approach into their teaching practice to help learners overcome their existing misconceptions and develop a deeper understanding of scientific concepts.

### **14. Conclusion**

In conclusion, this study provides valuable insights into the use of a conceptual change instructional strategy to improve learners' understanding of the quantitative aspects of chemical change. The study found that the conceptual change approach was an effective teaching strategy that led to a significant improvement in learners' scientific understanding after pre-test and post-test. The study also highlighted the importance of designing learning environments that allow learners to become aware of their current internal justifications and beliefs, which is essential for promoting conceptual change and improving problem-solving abilities.

The findings of the study have important implications for science education in South Africa and other countries facing similar challenges. Science educators should consider incorporating a conceptual change approach into their teaching practice to help learners overcome their existing misconceptions and develop a deeper understanding of scientific concepts. This approach has the potential to improve learners' academic performance and promote problem-solving abilities, which are critical skills for success in science and beyond. Overall, this study highlights the importance of adopting innovative teaching strategies that are grounded in educational research to promote effective learning and improve educational outcomes.

### **15. Recommendation**

Based on the findings of this study, there are several recommendations that can be made for science educators and policymakers. Firstly, science educators should consider incorporating a conceptual change approach into their teaching practice to help learners overcome their existing misconceptions and develop a deeper understanding of scientific concepts. This approach should be implemented in a well-designed learning environment that allows learners to reflect on their existing ideas and beliefs, which is essential for promoting conceptual change and improving problem-solving abilities. Secondly, policymakers should consider investing in teacher training and professional development programmes that focus on innovative teaching strategies that are grounded in educational research. These programmes can help to equip science teachers with the skills and knowledge they need to implement effective teaching strategies that promote conceptual change and improve educational outcomes. Lastly, further research should be conducted to explore the effectiveness of the conceptual change approach in other areas of science education and in other contexts. This can help to provide more evidence on the effectiveness of this approach and inform the development of more effective teaching strategies in science education.

*Implementation of Conceptual Change Approach to Improve Learners' Understanding… DOI: http://dx.doi.org/10.5772/intechopen.114094*


*2.1.5 Phage-colorimetric sensors*

Sensing based on changes in color allows the use of simple diagnostic systems like spectrophotometers, or even involving smartphones, and both of them are comparatively common and feasible. Designed colorimetric phage-based biosensors are mostly based and integrated on the utilization of reporter bacteriophages that carry genes coding for reporter enzymes. The foremost colorimetric sensor based on phage was to detect *Salmonella* ice nucleation sensor using reporter gene *inaW* [29]*.* Expression of ice nucleation protein was induced upon infection, interrupting the cell, and was consequently observed by the addition of an indicator dye (orange colored) [30]. Other serviceable reporter genes that have been successfully used with various colorimetric substrates are *celB* and *lacZ* segments encoding β-galactosidase and β-glycosidase [31]. More recently enhanced phage-based colorimetric technique has been reported to be integrating and coupling with novel technologies like surface plasmon [32], macroscope and smartphone [33], and lateral flow assay [11]. Other colorimetric phage-based biosensors established in recent years are briefed in **Table 1**.


*4.3. The Implications of Arable Land and Irrigation Water Resources Quota for Ecological Migration Policy*

Arable land and water resources remain the critical restrictive factors among natural resources necessary for agriculture, especially in arid areas. The relatively long history of voluntary out-migration in the study area, especially among the northern villages bordering desert, is driven mainly by declining arable land and irrigation water resources. The implementation of the river governance program in Shiyang River Basin has amplified the shortage of arable land and irrigation water resources quota. The remarkable diminution of arable land and water resources for agricultural production has resulted in immediate reduction of the natural capitals for the rural households' traditional livelihoods. To cope with these tensions, migration may be one of the adaptive livelihood strategies [15,35]. This viewpoint is backed by the out-migration history in the study area.

As the inland river governance planning has dual aims, to rehabilitate the eco-environment and to improve the victims' livelihood, the measures to decrease the arable land and irrigation water resources quota must be accompanied by measures to diversify livelihood and to develop water/land-saving industries. Otherwise, the migration intentions could not be transformed into actual migration actions, and the environmental conflicts might be transformed into social conflicts. The results of this study indicate that the respondents have more odds of intending to migrate when they have less arable land quota or when they deem water resources insufficient both for domestic use and agricultural production. A compulsory ecological migration policy is not recommended; with such a policy, the forced out-migration would be a failure of the social-ecological system to adapt as stated by Warner [35].


**3. Results**

#### **3.1 Antimicrobial susceptibility**

The 90 isolates showed resistance to penicillin (100%), oxacillin (99.3%), amoxicillin (100%) , cefotaxime (100%), cephalothin (100%), cefazolin (100%), chloramphenicol (100%), imipenem (99.3%), ciprofloxacin (87.7%); eleven strains (12.2%) showed low susceptibility for clindamycin, erythromycin, clarithromycin and were susceptible to ciprofloxacin; two strains (2.2%) showed low susceptibility for oxacillin (MIC 4µg/mL) and imipenem. All strains were susceptible to rifampin, tetracycline, gentamicin, trimethoprim-sulfamethoxazole and vancomycin.

Pulsed Field Gel Electrophoresis in Molecular Typing and

Epidemiological Detection of Methicillin Resistant *Staphylococcus aureus* (MRSA) 183

Fig. 2. Dendrogram comparing MRSA clones A, B and C from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico with different international MRSA clones: BK2464-New York/Japan-USA clone; JP27-New York/Japan-Japan clone; HDE288-Pediatric clone; HU25-Brazilian-clone; HPV107-Iberian clone; EMRSA-16-U.K. clone. For cluster analysis, Dice coefficients were calculated to compute matrix similarity a transformed into

an agglomerative cluster with the unweighted pair group method with arithmetic

Number of subtypes 5 3 6 SSC*mec* type3 IV II II

ST 30 305/36 5 1Antibiotic abbreviations: CLA- clarithromycin; CD – clindamycin; ERY – erythromycin GEN gentamicin; CIP - ciprofloxacin. 2Intermediate resistance pattern. 3Staphylococcal cassette chromosome

Table 1. Antibiotype and Genotypic characterization of the MRSA clones presented in the

(CLA,CD,ERY)2

*mec*. 4Except B1 and B2; 5Sequence typing (MLST), only the patterns B1 and B2.

Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico (2002-2009).

**Property Clone A Clone B Clone C**

ß-lactams, (CIP, CLA,CD, ERY)

˜180 ˜2114 ˜211

˜180 ˜211-640 ˜211-640

ß-lactams, (CIP, CLA,CD, ERY)

average.

*SmaI-mecA*

*Sma-Tn554*

Antibiotype1 (Resistance) ß-lactams,

Hybridization bands(Kb)

Hybridization bands(Kb)

#### **3.2 Molecular typing**

#### **3.2.1 PFGE analysis**

The PFGE analysis separated the MRSA strains into three types, A (5 subtypes), B (3 subtypes) and C (6 subtypes) (Figure 1). PFGE pattern C and subtypes were predominant in this isolates n=72 (80%), Clone A, n=11 (12.2%) and B, n=7 (7.8%) were only found in the isolates of 2002, and these two clones (A and B) were totally replaced by clone C in 2004 and continue until 2009. The results produced by a computer analysis of the banding patterns show clearly the division of the three clone groups (A, B and C); interestingly, the A and B clone isolates have very similar PFGE patterns (coefficient similarity 95%). Nevertheless, the three clones A, B and C could easily be distinguished by antibiograms and other molecular properties as well (Table 1). The three clones were multiresistant, however, each one of them showed a characteristic resistance pattern; clone A was resistant to ß-lactams and showed a low susceptibility to clarithromycin, clindamycin, erythromycin and was susceptible to ciprofloxacin; while clones B and C were resistant to ß-lactams, clarithromycin, clindamycin, erytromycin and ciprofloxacin; only the strains with subtypes B1 and B2 showed low susceptibility for oxacillin and imipenem.

Fig. 1. Pulsed field gel electrophoresis profiles of MRSA clinical isolates from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico and representatives of international clones. Lanes: 1-14 lambda ladder used a molecular size (MW) markers; 2 and 13 reference strain NCTC8325; 3-4 (44CAR and 47CAR) pattern C; 5 (2CAR) pattern A; 6 (20CAR) pattern B; 7-12 (HDE288, BK2464, JP27, EMRSA16, HPV107 and HU25) control strains representative of Pediatric, New York/Japan-USA, New York/Japan-Japan, EMRSA16-U.K, Iberian and Brazilian clones.

The PFGE analysis separated the MRSA strains into three types, A (5 subtypes), B (3 subtypes) and C (6 subtypes) (Figure 1). PFGE pattern C and subtypes were predominant in this isolates n=72 (80%), Clone A, n=11 (12.2%) and B, n=7 (7.8%) were only found in the isolates of 2002, and these two clones (A and B) were totally replaced by clone C in 2004 and continue until 2009. The results produced by a computer analysis of the banding patterns show clearly the division of the three clone groups (A, B and C); interestingly, the A and B clone isolates have very similar PFGE patterns (coefficient similarity 95%). Nevertheless, the three clones A, B and C could easily be distinguished by antibiograms and other molecular properties as well (Table 1). The three clones were multiresistant, however, each one of them showed a characteristic resistance pattern; clone A was resistant to ß-lactams and showed a low susceptibility to clarithromycin, clindamycin, erythromycin and was susceptible to ciprofloxacin; while clones B and C were resistant to ß-lactams, clarithromycin, clindamycin, erytromycin and ciprofloxacin; only the strains with subtypes B1 and B2 showed low

Fig. 1. Pulsed field gel electrophoresis profiles of MRSA clinical isolates from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico and representatives of international clones. Lanes: 1-14 lambda ladder used a molecular size (MW) markers; 2 and 13 reference strain NCTC8325; 3-4 (44CAR and 47CAR) pattern C; 5 (2CAR) pattern A; 6 (20CAR) pattern

representative of Pediatric, New York/Japan-USA, New York/Japan-Japan, EMRSA16-U.K,

B; 7-12 (HDE288, BK2464, JP27, EMRSA16, HPV107 and HU25) control strains

**3.2 Molecular typing 3.2.1 PFGE analysis**

susceptibility for oxacillin and imipenem.

Iberian and Brazilian clones.

Fig. 2. Dendrogram comparing MRSA clones A, B and C from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico with different international MRSA clones: BK2464-New York/Japan-USA clone; JP27-New York/Japan-Japan clone; HDE288-Pediatric clone; HU25-Brazilian-clone; HPV107-Iberian clone; EMRSA-16-U.K. clone. For cluster analysis, Dice coefficients were calculated to compute matrix similarity a transformed into an agglomerative cluster with the unweighted pair group method with arithmetic average.

1Antibiotic abbreviations: CLA- clarithromycin; CD – clindamycin; ERY – erythromycin GEN gentamicin; CIP - ciprofloxacin. 2Intermediate resistance pattern. 3Staphylococcal cassette chromosome *mec*. 4Except B1 and B2; 5Sequence typing (MLST), only the patterns B1 and B2.

Table 1. Antibiotype and Genotypic characterization of the MRSA clones presented in the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico (2002-2009).

Pulsed Field Gel Electrophoresis in Molecular Typing and

type 30 (ST30) .

(pattern B2).

(Figure 2).

**3.2.3 Homology pattern**

Epidemiological Detection of Methicillin Resistant *Staphylococcus aureus* (MRSA) 185

MRSA strains of clones B and C had SCC*mec* type II, sequences type 36 and 5 (ST36 and ST5) respectively, except B1 and B2 which did not amplify SCC*mec*, this isolates showed sequence

Fig. 4. (A) PFGE patterns of MRSA strains from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico. Lane 1, 3CAR, (pattern B1); lane 2, control strain and lane 3, 8CAR

One isolate belonging to each type of clones (A, B and C) were compared to strains belonging to previously characterized MRSA clones, i. e., representatives of the pediatric clone and isolates belonging to the New York-Japan clone and also to other international pandemic clones, namely, the Iberian, Brazilian and EMRSA-16 clones (Figure 1). Clone C showed a high degree of similarity to the pediatric (85.5%) and the New York-Japan (89.5%) clones. Clones A and B showed a high degree of similarity to the EMRSA-16 (80%) clone

#### **3.2.2 Hybridization pattern**

The hybridization patterns with *mecA* and Tn*554* probes indicated that the MRSA strains accompanying clone A carried the *mecA* gene on a *Sma*I fragment of approximately 180 Kb, while the *mecA* gene of the clones B and C were found on a fragment of approximately 211 Kb (Figure 3-A). One Tn*554* copy was identified usually on the fragment approximately 180 Kb between the isolates of clone A; while the MRSA strains accompanying clones B and C usually carried two identified Tn*554* copies between the *SmaI* fragments of approximately 211 and 640 Kb; only the strains 3CAR, (B1) and 8CAR, (B2), carried the transposon Tn*554* in a fragment of 640 Kb (Figure 3-B).

Fig. 3. (A) *SmaI*-*mecA* and (B) *SmaI* Tn*554* patterns identified among the clones A, B and C of the Instituto Nacional de Cardiología "Dr. Ignacio Chavéz, Mexico. Lane 1, molecular weight markers, lambda ladder; lane 2, 2CAR (pattern A); lane 3, 20CAR (pattern B) and lane 4, 44CAR (pattern C).

Two strains collected in this hospital 3CAR, (pattern B1) and 8CAR, (pattern B2) did not hybridize with the *mecA* DNA probe, interestingly both presented a low susceptibility to oxacillin and these isolates were subtypes of pattern B. The only difference was found in the *SmaI* hybridization fragment, which contains the *mecA* gene: in the two isolates, this fragment had a smaller molecular size (145 instead of 180 Kb) and did not react with the *mecA* probe, indicating a deletion of approximately 35 Kb, which must have included both the *mecA* gene and part of the *mec* element (Figure 4A and 4B). All the isolates accompanying clone A presented SCC*mec* type IV and sequence type 30 (ST30) whereas the MRSA strains of clones B and C had SCC*mec* type II, sequences type 36 and 5 (ST36 and ST5) respectively, except B1 and B2 which did not amplify SCC*mec*, this isolates showed sequence type 30 (ST30) .

Fig. 4. (A) PFGE patterns of MRSA strains from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico. Lane 1, 3CAR, (pattern B1); lane 2, control strain and lane 3, 8CAR (pattern B2).

## **3.2.3 Homology pattern**

184 Gel Electrophoresis – Advanced Techniques

The hybridization patterns with *mecA* and Tn*554* probes indicated that the MRSA strains accompanying clone A carried the *mecA* gene on a *Sma*I fragment of approximately 180 Kb, while the *mecA* gene of the clones B and C were found on a fragment of approximately 211 Kb (Figure 3-A). One Tn*554* copy was identified usually on the fragment approximately 180 Kb between the isolates of clone A; while the MRSA strains accompanying clones B and C usually carried two identified Tn*554* copies between the *SmaI* fragments of approximately 211 and 640 Kb; only the strains 3CAR, (B1) and 8CAR, (B2), carried the transposon Tn*554*

 Fig. 3. (A) *SmaI*-*mecA* and (B) *SmaI* Tn*554* patterns identified among the clones A, B and C of the Instituto Nacional de Cardiología "Dr. Ignacio Chavéz, Mexico. Lane 1, molecular weight markers, lambda ladder; lane 2, 2CAR (pattern A); lane 3, 20CAR (pattern B) and

Two strains collected in this hospital 3CAR, (pattern B1) and 8CAR, (pattern B2) did not hybridize with the *mecA* DNA probe, interestingly both presented a low susceptibility to oxacillin and these isolates were subtypes of pattern B. The only difference was found in the *SmaI* hybridization fragment, which contains the *mecA* gene: in the two isolates, this fragment had a smaller molecular size (145 instead of 180 Kb) and did not react with the *mecA* probe, indicating a deletion of approximately 35 Kb, which must have included both the *mecA* gene and part of the *mec* element (Figure 4A and 4B). All the isolates accompanying clone A presented SCC*mec* type IV and sequence type 30 (ST30) whereas the

**3.2.2 Hybridization pattern**

in a fragment of 640 Kb (Figure 3-B).

lane 4, 44CAR (pattern C).

One isolate belonging to each type of clones (A, B and C) were compared to strains belonging to previously characterized MRSA clones, i. e., representatives of the pediatric clone and isolates belonging to the New York-Japan clone and also to other international pandemic clones, namely, the Iberian, Brazilian and EMRSA-16 clones (Figure 1). Clone C showed a high degree of similarity to the pediatric (85.5%) and the New York-Japan (89.5%) clones. Clones A and B showed a high degree of similarity to the EMRSA-16 (80%) clone (Figure 2).

Pulsed Field Gel Electrophoresis in Molecular Typing and

**5. Conclusion**

MRSA clones.

**7. References**

**6. Acknowledgment**

We thank PhD. Lilia Chihu for style review of the paper

Epidemiological Detection of Methicillin Resistant *Staphylococcus aureus* (MRSA) 187

population, together with the few means of antibiotic restriction it could represent a potential short term risk for the VRSA appearance in the hospitals of our country. Clone A and B were only found in the isolates in 2002, these clones showed a high degree of similarity to the EMRSA-16 clone, this clone is one of the dominant types of MRSA found in a UK hospital (Moore & Lindsay, 2002) and was widely disseminated in Canada (Simor et al., 2002), Greece (Aires de Sousa et al., 2003) and Mexico (Aires de Sousa et al., 2001). Interestingly, both clones (A and B) are very similar (95%) (Figure 2), nevertheless, clone A showed a reduced resistance profile as clone B, and this is because of the existence of the SCC*mec* IV in these isolates, this chromosomal cassette was found in relation to isolated MRSA strains in the community (CA-MRSA) (Coombs et al., 2011). Different reports of several infections caused by CA-MRSA in Latin America (Uruguay, Rio de Janeiro, Colombia, Argentina and Mexico) have been published (Alvaréz et al., 2006; Ma et al., 2005; Reyes et al., 2009; Ribeiro et al., 2005; Velazquez et al., 2011). All pattern of PFGE of the clones A, B and C showed subtypes. Probably the PFGE subtypes indicate the continued

evolutionary divergence of these clones during its massive geographic expansion.

Relative genetic instability of the *mecA* element was observed in two strains and this was associated with an apparent deletion of the *mec* element, these isolates were very similar to profile B (B1 and B2) and presented a low susceptibility to oxacillin. In the literature there are reports of *S. aureus* strains with low-level methicillin resistance (MIC 2-4 µg/mL) which are not associated to the presence of *mecA* gene, Tomasz et al. reported one class of borderline methicillin-resistant strains having PBP1 and PBP2 with altered methicillinbinding affinities and overproduction of PBP4 (Tomasz et al., 1989). Another class of lowsusceptibility has been reported and was attributed to overproduction of penicillinase (McDougal & Thornsberry, 1986). Hackbarth et al. studied the nucleotide sequence of the PBP2 gene and identified a point mutation near the penicillin-binding motive of transpeptidase (Hackbarth et al., 1995). An MRSA clinical strain with significant methicillin resistance (MIC 64µg/mL) despite absence of *mec A* was reported (Yoshida et al., 2003).

The combination of molecular typing methods (PFGE, *mecA*, Tn*554* probes, SCC*mec*, and MLST) with epidemiologic and clinical information allows the detection of MRSA clusters and outbreaks and therefore provides a rationale for appropriate infection control intervention. Our study emphasizes the need of national and international collaborations to monitor the spread of current epidemic strains as well as the emergence of new ones in our country. The mechanisms of spread in different areas are poorly understood and further studies are necessary to understand the dynamics involved in the predominance of unique

Aires de Sousa, M. Miragaia, M. Santos, I. Avila, S. Adamson, I. Casagrande, S. Brandileone,

C. Palacio, R. Dell'Acqua, L. Hortal, M. Camou, T. Rossi, A. Velazquez, ME.