Genotypic analysis and antimicrobial
resistance of invasive Streptococcus pneumoniae serotype 14 by
Pulsed-Field Gel Electrophoresis in children under 5 years of age in Paraguay
María Eugenia León1,
Aníbal Kawabata1,
Helena Ortíz1,
Gustavo Chamorro1.
1Respiratory and Meningeal Diseases Section,
Central Public Health Laboratory
Asunción, Paraguay.
ABSTRACT
Introduction. Streptococcus pneumoniae serotype 14 is an important cause of invasive pneumococcal diseases throughout the world. It often presents resistance to a variety of antimicrobial agents, resulting in difficulties in treatment. Limited data are available on Streptococcus pneumoniae serotype 14 in the pediatric population in Paraguay, therefore it`s important monitoring of serotypes and antimicrobial resistance causing pneumococcal disease is important.
Objective: To evaluate the clonal relationships of isolates of invasive S. pneumoniae serotype 14 recovered from children under five years of age and to associate S. pneumoniae strains according to their antibiotic resistance.
Materials and methods. Forty-seven isolates with susceptibility data to penicillin, ceftriaxone, erythromycin, trimethoprim sulfamethoxazole, chloramphenicol, vancomycin and tetracycline, isolated from children under five years of age, were studied. The data was generated from strains collected in 2011-2013. The restriction pattern of the DNA was determined by pulsed field gel electrophoresis, using the enzyme SmaI. The genetic similarity between the isolates and the clone was established according to the Tenover criteria.
Results. Serotype 14 presented resistance to erythromycin 29.8%, trimethoprim-sulfamethoxazole 65.9% and tetracycline 42.5%, finding 11 isolates genetically related to the international clone England 14-9 with resistance to erythromycin.
Conclusion. These results provide information about the circulating clones in order to monitor the antibiotic resistance patterns. There is a need for better data on drug resistance in developing countries for characterizes the magnitude of the problem, provide effective interventions (conjugate vaccines), and antibiotic use control programs.
Keywords: Streptococcus pneumoniae, pulsed field gel electrophoresis, PFGE, antimicrobial resistance.
Artículo recibido: 27 marzo 2021
Aceptado para publicación: 30 abril 2021
Correspondencia: [email protected]
Conflictos de Interés: Ninguna que declarar
Análisis genotípico y resistencia antimicrobiana de Streptococcus pneumoniae serotipo 14 invasivo por electroforesis en gel de campo pulsado en niños menores de 5 años en Paraguay
RESUMEN
Introducción. El serotipo 14 de Streptococcus pneumoniae es una causa importante de enfermedades neumocócicas invasivas en todo el mundo. A menudo presenta resistencia a una variedad de agentes antimicrobianos, lo que genera dificultades en el tratamiento. Se dispone de datos limitados sobre Streptococcus pneumoniae serotipo 14 en la población pediátrica de Paraguay, por lo que es importante el monitoreo importante de los serotipos y la resistencia a los antimicrobianos que causan la enfermedad neumocócica.
Objetivo: Evaluar las relaciones clonales de aislamientos de S. pneumoniae invasivo serotipo 14 recuperados de niños menores de cinco años y asociar cepas de S. pneumoniae según su resistencia a antibióticos.
Materiales y métodos. Se estudiaron 47 aislamientos con datos de susceptibilidad a penicilina, ceftriaxona, eritromicina, trimetoprim sulfametoxazol, cloranfenicol, vancomicina y tetraciclina, aislados de niños menores de cinco años. Los datos se generaron a partir de cepas recolectadas en 2011-2013. El patrón de restricción del ADN se determinó mediante electroforesis en gel de campo pulsado, utilizando la enzima SmaI. La similitud genética entre los aislados y el clon se estableció de acuerdo con los criterios de Tenover.
Resultados. El serotipo 14 presentó resistencia a eritromicina 29,8%, trimetoprim-sulfametoxazol 65,9% y tetraciclina 42,5%, encontrándose 11 aislados genéticamente relacionados con el clon internacional England 14-9 con resistencia a eritromicina.
Conclusión. Estos resultados proporcionan información sobre los clones circulantes para controlar los patrones de resistencia a los antibióticos. Es necesario contar con mejores datos sobre la resistencia a los medicamentos en los países en desarrollo para caracterizar la magnitud del problema, proporcionar intervenciones efectivas (vacunas conjugadas) y programas de control del uso de antibióticos.
Palabras clave: Streptococcus pneumoniae, electroforesis en gel de campo pulsado, PFGE, resistencia a los antimicrobianos.
 |
INTRODUCTIONStreptococcus pneumoniae (pneumococcus) is the causative agent of serious invasive diseases such as pneumonia, meningitis and bacteremia and of non-invasive diseases such as otitis and sinusitis (1,2). Pneumococcal disease continues to be one of the leading causes of death among diseases prevented by vaccination according to the World Health Organization (WHO) (3). It is a pathogen associated with high morbidity and mortality worldwide, particularly in children and in countries with less access to diagnosis and treatment and whose importance is related to the burden of the disease, especially in the pediatric population, with the associated risks to the development of antimicrobial resistance and its therapeutic consequences (4). The polysaccharide capsule is considered the main determinant of virulence, protecting bacteria from phagocytosis (5). Isolates lacking the capsule almost never cause invasive disease. Based on structural differences in their capsular polysaccharides, 100 pneumococcal serotypes have been identified to date (6) but only a few are responsible for most invasive infections (7).
Some serotypes are more frequently associated with invasive diseases (8). Serotype 14 is one of the prevalent serotypes before the introduction of vaccines, which in addition to having a good capacity to colonize the nasopharynx of children, causes invasive pneumococcal diseases worldwide and often expresses resistance to a variety of antimicrobial agents, which include penicillin, erythromycin and ceftriaxone, which makes treatment difficult (9,10). The overall increase in penicillin resistance and multidrug resistance in pneumococci appears, in large part, as a result of the spread of highly resistant pneumococcal clones. For decades, pneumococci resistant to penicillin, erythromycin, and trimethoprim-sulfamethoxazole have spread rapidly worldwide. Resistances to tetracycline and chloramphenicol have also been identified, with rates that vary by region and population (11).In Paraguay, serotype 14 is the prevalent serotype in invasive diseases in children under five years of age. In 2011 a prevalence of 50.9% was observed, 36.4% in 2012 and 20.9% in 2013. In cases of meningitis, 54.8% were resistant to penicillin, 4% to Ceftriaxone, 21.4% to erythromycin and 59.5% to trimethoprim-sulfamethoxazole. In cases of non-meningeal infections, antimicrobial resistance is lower with 0.4% resistance to penicillin, 0.03% to ceftriaxone, 1.7% to erythromycin and 4.9% to trimethoprim-sulfamethoxazole (12-14).
The pulsed field gel electrophoresis (PFGE) technique has been widely applied and considered one of the main tools for epidemiological and surveillance studies (15,16). Due to its ability to differentiate isolates of the same species and relate them to endemic clones, it has also been one of the most frequent methods used for molecular typing of S. pneumoniae (17-19).
The objective of this study was to determine the clonal relationships of invasive isolates of S. pneumoniae serotype 14, recovered from children under five years of age and to associate strains of S. pneumoniae according to their antibiotic resistance.
Limited data are available on Streptococcus pneumoniae serotype 14 in the pediatric population in Paraguay, thus, there is a clear need for close monitoring of serotypes and antimicrobial resistance causing pneumococcal disease to contribute public health actions.
MATERIALS AND METHODS
Study population and samples
A descriptive, retrospective cross-sectional study that included strains of S. pneumoniae serotype 14 isolated in the period from January 1, 2011 to December 31, 2013.
A total of 47 invasive isolates of S. pneumoniae serotype 14 recovered from invasive disease in children under 5 years of age were studied during the period 2011 to 2013, through the surveillance program of the SIREVA II project of the Pan American Health Organization (PAHO), which were referred to the Central Laboratory of Public Health by laboratories of the health services of the country (Pediatric General Hospital, Institute of Social Security, Institute of Tropical Medicine, National Hospital of Itauguá, Regional Hospital Eastern City, San Lorenzo Maternal and Child Center, Institute of Respiratory and Environmental Diseases, Trauma Hospital, General Hospital Worker Neighborhood, Sanatorium Migone, Italian Sanatorium and Diaz Gill Laboratory).
Isolation and susceptibility
The isolates were identified by colony morphology, bile solubility tests, and optochin sensitivity. The isolates had Quellung capsular reaction serotype data (Statens Serum Institut, Copenhagen, Denmark). The antimicrobial susceptibility to penicillin (PEN) and ceftriaxone (CRO) were determined by Minimal inhibitory Concentration (MIC) and for erythromycin (ERY), chloramphenicol (CHL), trimethoprim-sulfamethoxazole (SXT), tetracycline ( TET), and vancomycin (VA) using the method disk diffusion (Kirby-Bauer), in accordance with regionally standardized techniques (20) and in accordance with the recommendations of the National Committee of Standards for the Clinical Laboratory (Clinical Laboratory Standards Institute, CLSI) (21).
Control strains of S. pneumoniae ATCC 700669 (clone Spain23F-1 ST81 with resistance to PEN, CHL, TET), ATCC 700671 (clone Spain9V-3, ST156 with resistance to PEN, SXT), ATCC 700676 (clone England14-9, ST9, resistant to ERY), ATCC 700677 (clone Slovakia14-10, ST20, resistant to PEN, ERY and TET), ATCC 700902 (clone Spain14-5, ST18 resistant to TET and CHL) and ATCC 49619 were included.
The strains were conserved in 20.0% skim milk powder (Oxoid, United Kingdom) with glycerol at ultra temperature -80ºC.
PFGE
Briefly, isolates were grown in supplemented Todd-Hewitt broth until they reached the log phase of growth. The cells were embedded in low melting point agarose discs (Bio-Rad) and digested with the restriction enzyme SmaI (Promega, United Kingdom) at a concentration of 20 U/mL; restriction fragments were separated on 1% agarose gels (Bio-Rad, USA) by pulsed field electrophoresis using the CHEFF II kit (Bio-Rad, USA).
Electrophoresis was carried out using a 1% PFGE agarose gel in a CHEF-DR III (Biorad System, USA) origin for 15 hours at 14ºC with 5 to 35 s of linear ramp at 6 V / cm and 120 ºC. The gels were stained with ethidium bromide and visualized with ultraviolet light. The PFGE genotype was defined as isolates showing> 90% identity in the dendrogram created using GelCompare II (Applied Maths, Kortrijik, Belgium).
DATA ANALYSIS
The data were collected and entered into a database in an Excel 2007 spreadsheet and analyzed using the statistical program Stata11.0 (Stata Statistical Software: Release 11. College Station, TX: StataCorp LP). The quantitative variables were expressed in measures of central tendency and measures of dispersion.
The distribution profiles of the PFGE electrophoretic run were compared taking into account the Tenover criteria (22), to determine the possible clonal groups (clusters) or distribution patterns of the Streptococcus pneumoniae strains (Serotype 14).
The electrophoretic patterns obtained were analyzed with the Gel Compare II software version 5.1 (Applied Maths, Kortrijik, Belgium), applying the Dice coefficient and the UPGMA method, with 1.5% tolerance and 1.5% optimization parameters for the analysis. It was carried out according to the protocol of Soares et al. (23) and the modifications of Vela et al. (24) as well as the elaboration of the homology dendrogram of the electrophoretic patterns.
Ethical aspects
This study was approved by the Research Ethics Committee of the Central Public Health Laboratory (CEI), resolution Nº. 35/280214. No informed consent statement was obtained because the study is observational, retrospective, with minimal or no risk for the subjects. The confidentiality of the data provided to the person was respected at all times and only the researchers were able to access the referred data.
RESULTS
In 2011, 29/59 (49.6%) cases of IPD were registered by serotype 14, in 2012 16/52 (30.7%) and in 2013 9/45 (20.0%). The general prevalence during the three years of study was 34.6%.
A total of 47 strains of S. pneumoniae isolated from invasive disease were studied in children under 5 years of age in Paraguay during the year 2011-2013. About half (55.3%) of the strains were isolated from female patients, 29.8% from children under 1 year, 72.3% of the cases corresponded to acute bacterial pneumonia and 80.8% were blood samples. Table 1.
Table 1. Socio-demographic characteristics of Streptococcus pneumoniae serotype 14 isolates from invasive disease in Paraguay (2011-2013) (n = 47).
|
Socio-demographic characteristics |
S.pneumoniae isolates |
% |
|
Gender |
||
|
Female |
26 |
55.3% |
|
Male |
21 |
45.7% |
|
Age |
||
|
< 1 year |
14 |
29.8% |
|
1 year |
9 |
19.1% |
|
2 years |
13 |
27.7% |
|
3 years |
7 |
14.9% |
|
4 years |
4 |
8.5% |
|
Diagnosis |
||
|
Meningitis |
3 |
6.4% |
|
Bacterial Pneumonia |
34 |
72.3% |
|
Community acquired pneumonia |
6 |
12.8% |
|
Sepsis |
4 |
8.5% |
|
Samples |
||
|
Cerebrospinal fluid |
3 |
6.4% |
|
Pleural fluid |
6 |
12.8% |
|
Blood |
38 |
80.8% |
The isolates studied from meningitis (MBA) 100% (3/3) were susceptible to penicillin G (PEN), ceftriaxone (CRO), chloramphenicol (CHL), erythromycin (ERY), trimethoprim-sulfamethoxazole (SXT), tetracycline (TET) and Vancomycin (VA). Isolates from bacterial pneumonia (NBA or NAC) 1/40 intermediate sensitivity to PEN and CRO (2.5%), 40/40 sensitive to CHL (100.0%), 12/40 resistant to ERY (30.0%), 29/40 resistant to SXT (72.5%), 19/40 resistant to TET (47.5%) and 40/40 sensitive to VA (100.0%). In sepsis isolates, 1/4 intermediate sensitivity to PEN (25.0%) and CTX, 4/4 sensitive to CHL (100.0%) and VA, 2/4 resistant to ERY and SXT (50.0%) and 1/3 resistant to TET (33.3%). Table 2. A general resistance to ERY of 29.8%, SXT 65.9% and TET 42.5% was found.
Table 2. Susceptibility profile of S. pneumoniae isolated in children under 5 years of age in Paraguay (2011-2013).
PEN1 Penicillin G
CRO2 Ceftriaxone
CHL3 Chloranphenicol
ERY4 Erythromycin
SXT5 Trimethoprim sulfamethoxazole
TET6 Tetracycline
VA7 Vancomycin
S8 Susceptible
I9 Intermediate
R10 Resistant
The analysis of the restricted products obtained by digesting the genome of the Streptococcus pneumoniae strains (Serotype 14) with SmaI, allowed the identification of 22 different patterns of bands (pulsetypes). The percentage of similarity ranged from 50.0% to 100.0% between isolates. Seven main clonal groups were identified, comprising A with 3 isolates (6.4%), B with 8 (17.0%), C with 2 isolates (4.2%), D with 11 isolates (23.4%), E with 2 isolates (4.2 %), F with 2 isolates (4.2 %), G with 5 isolates (10.6%) and 14 isolates were not genetically related.
Clonal groups A, B, C, E, F and G presented resistance related to TET and SXT. However, group E and F showed resistance only to SXT. In clonal group D all isolates show resistance to ERY. Figure 1. The isolates of the 7 clonal groups correspond to different health services in the country.
.
Figure 1. Dendrogram of Streptococcus pneumoniae serotype 14 strains by PFGE. Paraguay. 2011-2013
Eleven isolates of S. pneumoniae with antibiotic resistance to macrolides (ERY) were analyzed separately, resulting in a clonal group that according to Tenover's criteria are genetically indistinguishable from the England14-9 clone (ATCC 700676) (> 90% genetic relationship with clone England14-9 (10 S. pneumoniae isolates and 1 isolate with <90% similarity). Figure 2.
The only two isolates with intermediate resistance to the antibiotics PEN and CRO and resistance to SXT, whose electrophoretic profiles or pulsetypes show a difference of > 7 restriction fragments, were compared using PFGE, finding that they are not related to each other.
Figure 2. Analysis of macrolide-resistant S. pneumoniae clones compared to clone England 14-9 (n = 11).
DISCUSSION
In Paraguay, the prevalence of serotype 14 during the 2011-2013 period was 34.6%. According to the SIREVA II surveillance published by PAHO (12), the frequency of serotype 14 in 2011 in Argentina was 22.1%, Brazil 19.2% and Chile 31.9%. In Latin America, the most frequent serotype isolated in childhood disease was serotype 14 in 34.1% of the samples (95% CI: 29.4–38.9) in a study period of 2000-2016 (25). Serotype 14 was also found to be one of the most frequent in several studies conducted in countries such as the United States, China, Spain, and England (26-29).
The PCV10 vaccine was incorporated into the vaccination scheme of the Ministry of Public Health and Social Welfare of Paraguay in 2012, with serotype 14 found in a high frequency during the study period, mainly in children under 5 years of age (30). Several studies conducted with isolates of S. pneumoniae have suggested that serotype 14 is among the most important capsular types that drive the emergence of resistance to penicillin and other antibiotics (31-33).
The resistance found in this study was ERY 29.8%, SXT 65.9% and TET 42.5%, in addition to 2 isolates with intermediate sensitivity to PEN and CRO. Other authors in Brazil evaluated the susceptibility profile of S. pneumoniae isolated from invasive disease over a period of 16 years, finding the highest frequency of resistance (4.5%) in serotype 14 and in another study the highest rate of no susceptibility to SXT (51.2%), non-penicillin-sensitive pneumococci accounted for 27.3% of the isolates (MIC 0.12 to 4 μg / mL). Non-susceptibility to penicillin was strongly associated with serotypes 14 and 23F (34, 35). In Colombia, serotype 14 was the most prevalent in children under 2 years of age and between 2 and 5 years of age. In contrast, serotype 14 was related to penicillin resistance in 60.0%. Resistance to ERY increased from 7.6% (2009) to 9.8% (2010) (p = 0.17), associated with serotypes 6A (21.1%), 19A (20.3%), 6B (19.5%), 14 (13.0%) and other serotypes (n = 12) (26.1%) (36). Penicillin-resistant strains of S. pneumoniae serotype 14 have also been seen in countries such as Korea, Japan, and Morocco (37-39).
Taking into account that nasopharyngeal colonization is the gateway to pneumococcal disease and that the distribution of antimicrobial resistance patterns in nasopharyngeal S. pneumoniae carrier strains can predict resistance rates in invasive strains, in Russia high rates of resistance to penicillin accompanied by ERY and clindamycin were observed, and this may be due to an expansion of the endemic multi-resistance clone ST143 with serotype 14 (40). Another study carried out in 9 European countries showed the highest proportion of resistance to ceftazidime and penicillin among strains of serotype 14 (13 of 16, 81.3%) (41).
Molecular typing studies have identified multi-drug resistant S. pneumoniae clones, some of which are globally dispersed. Internationally disseminated antimicrobial resistant clones are Spain14–5 (ST18), England14–9 (ST9), CSR14–10 (ST20), Tennessee14–18 (ST67), Denmark14–32 (ST230) and Holland14–35 (ST124) according to the Pneumococcal Molecular Epidemiology Network (PMEN) (http://www.sph.emory.edu/PMEN/index.html).
In this study we detected a correlation between clonal groups of PFGE and resistance to antibiotics. The typing of the 47 strains of S. pneumoniae belonging to serotype 14 revealed 5 clonal groups, where most of the strains belonged to group B with 13 isolates with resistance profile to TET and SXT. Clonal groups B and C presented resistance related to TET and SXT, clonal group D showed resistance to ERY and clonal group E presented resistance to SXT.
Previous studies demonstrated the presence of 3 of these global clones: England14-9, Columbia23F-26 and Spain9V-3 within the group of resistant PEN isolates. Resistance to ERY was found mainly among PEN sensitive isolates where most were assigned to clone England14-9 (ST-15), a clone related to isolates serotype 14 (42). In Brazil, four important clonal complexes (CC) were shown to be responsible for the emergence and spread of antimicrobial resistance among S. pneumoniae strains of serotypes 14 and 9, three are related to PMEN clones, including CC156 (related to Spain9V-3), CC66 (related to Tennessee14-18) and CC15 (related to England14-9), while the fourth, CC5401, is characterized as a regional clone (43).
SXT is one of the antibiotics used for the treatment of S. pneumoniae infections (44). A group of researchers in Thailand found the highest rate of resistance to SXT (78.94%) of S. pneumoniae isolated from children (45).
Resistance to macrolides is observed with great variations between the range of <10 to> 90% according to geographic regions (46). In agreement with the study by Corso et al. Regarding invasive infections, the emergence of ERY-resistant S. pneumoniae was mainly due to England14-9, Poland 6B-20 and Spain 9V-3 international clones (47).
A clonal group resulted from 11 isolates of S. pneumoniae with resistance to macrolides, specifically to ERY, in which 5 of them obtained 100% clonality with the clone England14-9, other 5 isolates obtained >90% clonality and 1 isolation <90%. However, to confirm the results of this international comparison, it is required to use the multilocus sequence typing technique (MLST) with the isolates. The England14-9 clonal complex generally belongs to serotype 14 and is typically resistant to ERY, but sensitive to TET and CHL (9). In a study carried out in Brazil, they found 2 strains of S. pneumoniae belonging to serotype 14 resistant to erythromycin, cloned with the clone England14-9 but sensitive to PEN (48). Canadian S. pneumoniae isolates studied between 2007 and 2013 were related (at least five out of seven MLST allele numbers in common) to Spain23F-1 (ST81), Spain9V-3 (ST156), England14-9 (ST9), Taiwan19F -14 (ST236) and Sweden 15A-25 (ST63) (49).
The high rate of resistance can be caused by the indiscriminate use of antibiotics, the presence of a particular genotype, vaccination rates, and the presence of multi-resistant clones (50). The increase in resistance of S. pneumoniae to antibiotics such as penicillin, macrolides and trimethoprim-sulfamethoxazole is a worldwide concern, therefore it is relevant to control this resistance through strategies such as surveillance of circulating clones in order to monitor the resistance patterns and thus allow the proper use of antimicrobials that reduce the number of patients with invasive diseases.
Funding information
This work was supported by grants from the Mercosur Structural Convergence Fund (FOCEM) - Mercosur, FOCEM agreement N° 03/11 Project "Research, Education and Biotechnologies Applied to Health (COF 03/11).
Conflicts of interest
The authors declare that there no conflicts of interest.
Authors and contributors
|
Nombre |
ORCID |
Roles |
|
León Ayala María Eugenia |
0000-0001-7416-249X |
Conceptualization and Methodology Investigation and Supervision Validation and Writing – Review and Editing |
|
Kawabata Anibal |
0000-0002-3960-435X |
Conceptualization and Methodology Data Curation and Formal Analysis Investigation and Validation Writing - Review & Editing |
|
Helena Ortiz |
0000-0001-5617-1528 |
Supervision |
|
Chamorro Cortesi Gustavo |
0000-0003-3959-1533 |
Supervision and Project Administration Writing –Review and Editing |
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