Do Different Proportions of Antibiotics in the CTZ Paste Interfere with the Antimicrobial Action? In Vitro Study

Abstract

Objective:

To evaluate the antimicrobial action of the CTZ paste in three different proportions by diffusion in agar with the microorganisms: Enterococcus faecalis,Escherichia coli, and Candida albicans.

Material and Methods:

Three different proportions of antibiotics were tested: GROUP A - CTZ paste in the ratio of 33.33% chloramphenicol + 33.33% tetracycline + 33.33% zinc oxide, mixed with 2 drops of eugenol (1:1:1 ratio); GROUP B - CTZ paste in the proportion of 25% chloramphenicol + 25% tetracycline + 50% zinc oxide, mixed with 2 drops of eugenol (1: 1: 2 ratio); GROUP C - CTZ paste with 13% chloramphenicol + 13% tetracycline + 74% Zinc Oxide, mixed with 2 drops of eugenol (1:1:6 ratio); PC GROUP - Positive Control (0.12% Chlorhexidine); and NC GROUP - Negative Control (0.9% Saline solution). Data were analyzed through descriptive statistics (means and standard deviation). The one-way ANOVA and Tukey’s test were used, with a significance level of 5%

Results:

No statistical differences for Enterococcus faecalis between groups A, B, and C (p = 0.1986) were found. There were statistical differences for Escherichia coli between groups B and C (p = 0.029), and for Candida albicans between groups A and C (p = 0.006). Groups A, B, and C had significant differences with both Positive and Negative Controls for all the microorganisms

Conclusion:

The three different ratios of CTZ paste showed antimicrobial efficacy against Enterococcus faecalis,Escherichia coli, and Candida albicans microorganisms.

Keywords:
Tooth, Deciduous; Endodontics; Pulpectomy; Anti-Bacterial Agents

Introduction

The endodontic technique using a paste based on chloramphenicol, tetracycline, zinc oxide, and eugenol, referred to as CTZ, was introduced in the pediatric dentistry practice by Cappiello in 1964 ^[1]. It is an alternative for the endodontic treatment of deciduous teeth, whose technique is simplified and easy to perform, dispensing with the instrumentation of root canal systems, in addition to the possibility of being used in public health ^[1,2]. The success of the paste is due to its composition, since chloramphenicol and tetracycline are broad-spectrum antibiotics, and zinc oxide and eugenol have antimicrobial action ^[3].

The root canal system of the deciduous teeth has severe curvatures and many accessory canals. In addition, the deciduous teeth undergo rhizolysis in different directions and proportions ^[4]. Thus, instrumentation for the elimination of microorganisms is hampered. The use of antimicrobial pulps represents one of the most important aspects of treatment success ^[3].

In vitro studies have shown that the CTZ paste has higher antimicrobial activity compared to the Guedes-Pinto paste ^[3,5], zinc oxide and eugenol, calcium hydroxide paste and Vitapex^{® [5]}, Calen^®, L&C^®, zinc oxide, and eugenol and MTA ^[3]. In clinical practice, the use of CTZ paste in necrotic primary molars showed promising clinical and radiographic results ^[6]. In most of the studies, CTZ is used in a 1:1:2 ratio, 25% of chloramphenicol, 25% of tetracycline and 50% of zinc oxide, mixed with Eugenol ^[1,5-12].

Although the American Academy of Pediatric Dentistry (AAPD) ^[13] reports the increased prevalence of antibiotic-resistant microorganisms, no studies have been found in the literature evaluating the antimicrobial capacity of the CTZ paste with a decrease in the proportion of antibiotic. Thus, this work evaluated the in vitro antimicrobial action of the CTZ paste in three different proportions.

Material and Methods

Study Design

An in vitro study was carried out to evaluate the antimicrobial action of the CTZ paste in three different proportions by means of agar diffusion. Three microorganisms were used: Enterococcus faecalis,Escherichia coli, and Candida albicans. All these strains were isolated, identified, cultivated, and maintained viable in the laboratory of the São Leopoldo Mandic Research Center, Campinas, SP, Brazil.

The groups tested were: Group A - CTZ paste in the proportion of 33.33% chloramphenicol + 33.33% tetracycline + 33.33% zinc oxide, mixed with two drops of eugenol (1:1:1 ratio); Group B - CTZ paste in the proportion of 25% chloramphenicol + 25% tetracycline + 50% zinc oxide, mixed with 2 drops of eugenol (1:1:2 ratio); Group C - CTZ paste with 13% chloramphenicol + 13% tetracycline + 74% zinc oxide, mixed with 2 drops of eugenol (1:1:6 ratio); PC Group (Positive Control) - 0.12% Chlorhexidine Gluconate; and NC Group (Negative Control) - 0.9% Sterile saline solution (Table 1). The CTZ capsules were manipulated at the Fórmula & Ação Laboratory (São Paulo, SP, Brazil), in the amount of nine 200 mg capsules for group. All the manipulated capsules were mixed with 2 drops of Eugenol (Biodinâmica Química e Farmacêutica Ltda, Ibiporã, PR, Brazil).

Table 1
CTZ composition of groups A, B, and C, and substances used as positive (PC) and negative controls (NC).

Groups	Chloramphenicol	Tetracycline	Zinc Oxide	Eugenol	Ratio
A	33.33%	33.33%	33.33%	2 drops	1 : 1 : 1
B	25%	25%	50%	2 drops	1 : 1 : 2
C	13%	13%	74%	2 drops	1 : 1 : 6
PC	0.12% Chlorhexidine Gluconate
NC	0.9% Saline Solution

Agar Preparation

Nine Petri dishes were used, six of them containing 20 ml of the Brain Heart Infusion Agar medium (BHIA; Difco Laboratories, Detroit, MI, USA, lot 0000237071) for E. faecalis and E. coli, and the three remaining ones with the Sabouraud Dextrose Agar culture medium (Neogen Corporation, Lansing, MI, USA, lot 107876B) for C. albicans. After being autoclaved (at 121ºC with 1 ATM during 20 minutes), the plates were cooled, with the culture medium presenting 5mm thickness. The microbial mixture was incubated for 24 hours at 37ºC.

After this period, the cells were suspended in sterile saline solution (0.9%) (Dinâmica Química Contemporânea Ltda., Diadema, SP, Brazil, lot 68837). The suspension of each microbial strain was adjusted to match the tube of 1 McFarland turbidity standard. In the preparation of the microbial mixture, an inoculum of 200 μl of Enterococcus faecalis, of 400 μl of Escherichia coli and of 300 μl of Candida albicans was removed from each suspension and transferred to each sterile test tube, obtaining the test mixtures, which were used immediately after preparation. The plates were inoculated with the microbial suspension using disposable and sterile swabs (Absorve, CRAL Artigos para Laboratório LTDA, São Paulo, SP, Brazil). Three wells of 5 mm in diameter and 5 mm deep were used to insert the CTZ of groups A, B and C and an absorbent paper disc of the same diameter for the positive and negative controls in each Petri dish (Prolab, José dos Pinhais, PR, Brazil).

The capsules, previously encoded per group, were shaken and opened. For manipulation, the powder was dispensed into a sterile glass plate, and then two drops of Eugenol were added, with the glass in a vertical position. They were spatulated with a no. 24 spatula (SSWhite Duflex, Juiz de Fora, MG, Brazil) sterile, in the consistency of toothpaste for the same operator.

Soon after the paste was manipulated, it was placed in one of the three 5mm diameter wells of each Petri dish. After the wells were completely filled with the CTZ paste and the control groups, the plates were transferred to a bacteriological oven and kept in the incubator at 37 ºC for 24 hours.

Evaluation of Samples

Measurements of the diameters of the zones of inhibition of microbial growth were performed using a millimeter pachymeter (Mitutoyo Sul Americana Ltda., Suzano, SP, Brazil). The positive control was verified with the use of 0.12% chlorhexidine gluconate substance (Periogard, Colgate-Palmolive Indústria Ltda, São Bernardo do Campo, SP, Brazil) and the negative control with 0.9% sterile saline solution. During all phases of the experiment, the aseptic technique was observed, and the tests were conducted by the same operator. The evaluator was blind.

Data Analysis

For the analysis of the data, means and standard deviation of the inhibition zone size were used. One-way ANOVA and Tukey test were used with a significance level of 5%. IBM SPSS Statistics for Windows Software, version 25 (IBM, Armonk, NY, USA).

Results

The results of the halo diameters, in millimeters, after 24 hours of inhibition of bacterial growth induced by different concentrations of the CTZ paste in different proportions, that is, Group A (1: 1: 1), Group B (1: 1: 2), Group C (1: 1: 6), PC Group: 0.12% Chlorhexidine gluconate; NC Group: 0.9% Saline solution, are summarized in Table 2 and Figure 1 to 3, according to the microorganisms Enterococcus faecalis,Escherichia coli and Candida albicans, respectively.

Table 2
Measurement of the inhibition halo (mm) in groups.

Groups	E. faecalis	E. coli	C. albicans
	Mean (SD)	Mean (SD)	Mean (SD)
A	42.41 ± 1.17	40.16 ± 1.38	32.24 ± 1.12
B	41.55 ± 1.43	41.03 ± 0.41	29.94 ± 0.88
C	40.19 ± 1.36	38.38 ± 0.59	28.61 ± 0.49
PC	19.61 ± 0.64	20.42 ± 0.26	7.62 ± 2.19
NC	0	0	0

• *Groups A = CTZ 1:1:1 ratio; B= CTZ 1:1:2 ratio, C= CTZ 1:1:6 ratio, PC = 0.12% Chlorhexidine gluconate; NC = 0.9% saline solution.

Figure 1
Mean and standard deviation of the inhibition halos of the Enterococcus faecalis samples.

*Different letters indicate statistical differences between groups (p<0.05).

Figure 2
Mean and standard deviation of the inhibition halos of the Escherichia coli samples.

*Different letters indicate statistical differences between groups (p<0.05).

Figure 3
Mean and standard deviation of the inhibition halos of the Candida albicans samples.

*Different letters indicate statistical differences between groups (p<0.05).

The one-way analysis of variance (ANOVA) showed a statistical difference (p<0.05) in the groups tested regarding the size of the inhibition halo. The Tukey test showed that for the Enterococcus faecalis bacterium there was no statistical difference between groups A (42.41 ± 1.17), B (41.55 ± 1.43) and C (40.19 ± 1.36) (p<0.05) (Figure 1). For all microorganisms, there were statistical differences between all groups tested and the PC and NC, presenting a greater inhibition halo (Figure 1, 2 and 3).

Discussion

The root canal system of necrotic primary teeth has a polymicrobial nature, which is similar to that of permanent teeth, with a predominance of anaerobic microorganisms ^[14-17]. The present study tested Gram-negative (E. coli), Gram-positive (E. faecallis), both anaerobic, and fungus C. albicans.

The antimicrobial activity of the root canal system of the deciduous tooth with pulp necrosis is a characteristic of fundamental importance for the success of endodontic treatment ^[5,18]. Some studies reported a greater success of the CTZ paste in relation to the antimicrobial activity when compared to the other materials tested ^[3,5], proving to be an appropriate methodology to measure the antimicrobial potential of endodontic materials. However, there are no studies in the literature that have compared different proportions of antibiotics in the CTZ paste, showing that this is an unprecedented result, which makes it impossible to make more specific comparisons with our study. Despite this, all the proportions of the CTZ paste showed antimicrobial efficacy against the E. coli,E. faecalli, and C. albicans microorganisms.

For Enterococcus faecalis, the three paste proportions did not present significant differences, for Escherichia coli. There was no significant difference between Groups A and C and for Candida albicans. There was no significant difference between Groups B and C. All paste proportions presented an inhibition halo, which may suggest efficacy in the antimicrobial action. The mixture of zinc oxide and Eugenol has neutral pH, so it does not interfere with the action of the antibiotics tetracycline and chloramphenicol present in the CTZ paste. Group C, even with reduced amounts of antibiotics, showed sufficient and effective antimicrobial properties.

It is essential to know the pathogenic microbiota present in the root canal system to develop an effective endodontic therapy strategy. Enterococcus faecalis is found to be highly prevalent in root canals of endodontically treated teeth with persistent infection or secondary infections. It plays a crucial role in endodontic treatment failure due to its resistance and its ability to survive treatment. In addition, it can colonize areas inaccessible to disinfection mechanisms ^[19,20]. There was no significant difference between the groups A, B, and the experimental paste (Group C) in relation to the size of the inhibition halo against E. faecalis.

The rational use of antibiotics can be defined as the practice of prescription, which results in optimal indication, dosage, route of administration, and duration of a therapeutic or prophylactic regimen. It provides clinical success with minimal toxicity and reduces the impact on microbial resistance ^[21]. The indiscriminate use of antibiotics is considered by the World Health Organization (WHO) to be a global problem and, as a consequence, has led to the growth of bacterial resistance ^[22] and the emergence of superbugs ^[23]. The evaluation of the CTZ paste with a reduced concentration of chloramphenicol and tetracycline (Group C) aimed to promote the rational use of medications, justified by the fact that children's organs and tissues are developing and are more susceptible to the adverse effects of medication. And the response to medications is conditioned by factors such as age, size, body weight, stage of development, nutritional status, concomitant administration of other drugs, time of administration and pre-existing disease ^[24].

The results obtained in vitro by the experimental paste (Group C), with a reduction of the concentration of chloramphenicol and tetracycline, suggest that the dose of antibiotic of CTZ paste proposed by Cappiello in 1964 ^[1], is greater than that required for the endodontic treatment of bacteria present in the root canal system of deciduous teeth.

One of the issues related to the endodontic treatment of deciduous teeth is a color change. Thus, the disadvantage of the CTZ paste is the pigmentation of the dental crown caused by its components ^[8]. This fact generates resistance of pediatric dentists in the use of the CTZ. As the CTZ paste used in the experimental group (Group C) has a lower concentration of tetracycline, the main cause of color change, one possibility is that there is less color change. However, studies that evaluate this in the new proportion need to be performed.

The lack of scientific evidence on the biocompatibility of the CTZ paste and its influence on tooth enamel formation of the permanent successor germ has made its use not recommended in some countries. Some authors ^[25] observed different staining in 11.7% of the permanent successors of primary teeth treated with CTZ paste at 1:1:1 concentration (Group A). Due to the fact that staining was also observed in permanent teeth whose primary teeth had been treated with Formocresol, they suggested that this color change might be related to local periapical inflammation rather than the components of the formulas. Depending on the degree and severity of infection of the deciduous tooth, the permanent successor may exhibit structural abnormalities with alteration in enamel matrix formation (hypoplasia) or a deficiency in mineralization, altering enamel color (hypocalcification) ^[26].

In vitro results are very interesting and promising, and it is important to evaluate other properties, such as clinical efficacy and color change of tooth crown treated with CTZ paste in the experimental ratio of chloramphenicol and tetracycline.

Conclusion

The lower proportion of antibiotics in the CTZ paste did not interfere with the antimicrobial activity. The three different ratios of CTZ paste showed antimicrobial efficacy against E. coli,E. faecallis, and C. albicans.

• Financial Support: None.

Acknowledgments: The authors wish to thank Gilca Lacerda and Thiago Santos Almeida for collaboration in the microbiology laboratory of the São Leopoldo Mandic Research Center and Victor Angelo Martins Montalli for the data analysis.

References

• ^[1]
  Cappiello J. Tratamentos pulpares em incisivos primários. Rev Assoc Odontol Argentina 1964; 52:139-45.
• ^[2]
  Reis BS, Soares LC, Marques MM, Brum SC, Barbosa OLC, Barbosa CC. Antimicrobial activity CTZ front of bacteria. Braz J Surg Clin Res 2016; 16(3):58-61.
• ^[3]
  Piva F, Faraco-Junior IM, Feldens CA, Estrela CRA. Antimicrobial action of root canal filling materials for primary teeth using the agar diffusion method: An in vitro study. Pesqui Bras Odontopediatria Clín Integr 2009; 9(1):13-7. https://doi.org/10.4034/1519.0501.2009.0091.0003
  » https://doi.org/10.4034/1519.0501.2009.0091.0003
• ^[4]
  Sheffer OL, Storrer DLM, Lopes MN. Avaliação da possibilidade do preparo biomecânico dos condutos radiculares em molares decíduos. Dens 1973; 1:147-53. https://doi.org/10.5380/rd.v1i5.12160 [In Portuguese]
  » https://doi.org/10.5380/rd.v1i5.12160
• ^[5]
  Amorim LFG, Toledo OA, Estrela CRA, Decurcio DA, Estrela C. Antimicrobial analysis of different root canal filling pastes used in pediatric dentistry by two experimental methods. Braz Dent J 2006; 17(4):317-22. https://doi.org/10.1590/S0103-64402006000400010
  » https://doi.org/10.1590/S0103-64402006000400010
• ^[6]
  Moura LFAD, Lima MDM. Lima CCB, Machado JIAG, Moura MS, Carvalho, PV. Endodontic treatment of primary molars with antibiotic paste: A report of 38 cases. J Clin Pediatr Dent 2016; 40(3):175-7. https://doi.org/10.1590/S0103-64402006000400010
  » https://doi.org/10.1590/S0103-64402006000400010
• ^[7]
  Bruno GB, Alves APN, Menezes VA, Maia MCG, Bruno AF, Viana GSB. Biocompatibility evaluation of an antibiotic paste after pulpotomy in dogs. Braz J Oral Sci 2007; 6(22):1397-1401. https://doi.org/10.20396/bjos.v6i22.8642999
  » https://doi.org/10.20396/bjos.v6i22.8642999
• ^[8]
  González-Núñez D, Trejo-Quiroz P, De León-Torres C, Carmona-Ruiz D. Técnica de endodoncia no instrumentada mediante el uso de la pasta CTZ. Rev Estomat 2010; 18(2):27-32. [In Spanish]
• ^[9]
  Calixto-Chianca KS, Correa-Olaya AI, Anchelia-Ramirez SH. Effectividade clínica y radiográfica de dos pastas antibióticas empleadas en necrosis pulpar en ninos de um hospital nacional del Perú. KIRU 2014; 11(2):115-22. [In Spanish]
• ^[10]
  Siegl RMC, Lenzi TL, Politano GT, Benedetto M, Imparato JCP, Pinheiro SL. Two endodontics techniques analysis in primary molars with fistula. RGO 2015; 63(2)187-94. https://doi.org/10.1590/1981-863720150002000082990
  » https://doi.org/10.1590/1981-863720150002000082990
• ^[11]
  Lima CCB, Conde Júnior AM, Rizzo MS, Moura RD, Lima MDM, Moura LFAD. Biocompatibility of root filling pastes used in primary teeth. Int Endod J 2015; 48(5):405-16. https://doi.org/10.1111/iej.12328
  » https://doi.org/10.1111/iej.12328
• ^[12]
  Ferreira JL, Medina AR, Montoya MEH, Rosas CYD, Medrano LEC, Garcia IT. Efectividad clínica y radiográfica de la pasta antibiótica CTZ en pulpotomías de molares primarios. Ensayo clínico aleatorio controlado. Int J Odontostomat 2016; 10(3):425-31. https://doi.org/10.4067/S0718-381X2016000300008
  » https://doi.org/10.4067/S0718-381X2016000300008
• ^[13]
  Guideline on use of antibiotic therapy for pediatric dental patients. Pediatr Dent 2016; 38(6):325-7.
• ^[14]
  Toyoshima Y, Fukushima H, Inoue JI, Sasaki Y, Yamamoto K, Katao H, et al. A bacteriological study of periapical pathosis on deciduous teeth. JPN J Pedod 1988; 26(3):449-58. https://doi.org/10.11411/jspd1963.26.3_449
  » https://doi.org/10.11411/jspd1963.26.3_449
• ^[15]
  Sato T, Hoshino E, Uematsu H, Noda T. Predominant obligate anaerobes in necrotic pulp of human deciduous teeth. Microb Ecol Health Dis 1993; 6(6):269-75. https://doi.org/10.3109/08910609309141335
  » https://doi.org/10.3109/08910609309141335
• ^[16]
  Pazelli LC, Freitas AC, Ito IY, Souza-Gugelmin MCM, Medeiros AS, Nelson-Filho P. Prevalence of microorganisms in root canals of human deciduous teeth with necrotic pulp and chronic periapical lesions. Pesqui Odontol Bras 2003; 17(4):367-71. https://doi.org/10.1590/S1517-74912003000400013
  » https://doi.org/10.1590/S1517-74912003000400013
• ^[17]
  Silva LAB, Nelson-Filho P, Faria G, Souza-Gugelmin MCM, Ito IY. Bacterial profile in primary teeth with necrotic pulp and periapical lesions. Braz Dent J 2006; 17(2):144-8. https://doi.org/10.1590/S0103-64402006000200012
  » https://doi.org/10.1590/S0103-64402006000200012
• ^[18]
  Queiroz AM, Arid J, Nelson-Filho P, Lucisano MP, Silva RAB, Sorgi CA, et al. Correlation between bacterial endotoxin levels in root canals of primary teeth and the periapical lesion area. J Dent Child 2016; 83(1):9-15.
• ^[19]
  Nacif MCAM, Alves FRF. Enterococcus faecalis in endodontics: A challenge to success. Rev Bras Odontol 2010; 67(2):209-14.
• ^[20]
  Zoletti GO, Pereira EM, Schuenck RP, Teixeira LM, Siqueira Jr JF, Santos KRN. Characterization of virulence factors and clonal diversity of Enterococcus faecalis isolates from treated dental root canals. Res Microbiol 2011; 162(2):151-8. https://doi.org/10.1016/j.resmic.2010.09.018
  » https://doi.org/10.1016/j.resmic.2010.09.018
• ^[21]
  Dellit TH, Owens RC, McGowan JE Jr, Gerding DN, Weinstein RA, Burke JP, et al. Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America guidelines for developing an institutional program to enhance antimicrobial steward ship. Clin Infec Dis 2007; 44(2):159-77. https://doi.org/10.1086/510393
  » https://doi.org/10.1086/510393
• ^[22]
  Sampaio PS, Sancho LG, Lago RF. Implementation of new regulations for prescribing and dispensing of antibioticschallenges and possibilities. Cad Saúde Colet 2018; 26(1):15-22. https://doi.org/10.1590/1414-462x201800010185
  » https://doi.org/10.1590/1414-462x201800010185
• ^[23]
  Martins GS, Mangiacacchi BM, Borges FV, Lima NB. Uso indiscriminado de antibióticos pela população de São José do Calçado (ES) e o perigo das superbactérias. Acta Bio Bras 2015; 6(2):84-96. https://doi.org/10.18571/acbm.089
  » https://doi.org/10.18571/acbm.089
• ^[24]
  Pereira AC, Silveira VAS, Rosa LEB, Rocha RF. Drugs prescription in pediatric dentistry. Rev Odontol UNESP 2009; 38(4)256-62.
• ^[25]
  Santos-Júnior VE, Alencar Filho AV, Leite ACGL, Rosemblatt A. Is there an association between enamel stains in premolars and endodontic treatment of their primary molar predecessors? Pesqui Bras Odontopediatria Clín Integr 2013; 13(1):17-21. https://doi.org/10.4034/PBOCI.2013.131.03
  » https://doi.org/10.4034/PBOCI.2013.131.03
• ^[26]
  Corrêa FNP, Corrêa JPNP, Pellegrinetti MB, Imparato JCP. Endodontic treatment in predecessors of Turner tooth. Rev Inst Ciênc Saúde 2008; 26(2):258-62.

Edited by

Academic Editors: Alessandro Leite Cavalcanti and Wilton Wilney Nascimento Padilha

Publication Dates

• Publication in this collection
  13 Jan 2020
• Date of issue
  2019

History

• Received
  02 Apr 2019
• Accepted
  05 July 2019
• Published
  26 July 2019