Antimicrobial susceptibility of Streptococcus pneumoniae isolated from patients in the northeastern macroregion of São Paulo state, Brazil, 1998-2013

Introduction: There are reports worldwide about the increase in infections caused by Streptococcus pneumoniae resistant to antimicrobials. Objective: Evaluate the susceptibility profile of serotypes of Streptococcus pneumoniae associating them with pneumococcal invasive diseases (PID), as well as antimicrobial therapies. Method: This is a retrospective cross-sectional research involving secondary data from 1998 to 2013, in the northeastern macroregion of São Paulo state, Brazil, composed of Araraquara, Barretos, Franca and Ribeirão Preto regions, with 90 municipalities. At Instituto Adolfo Lutz (IAL), isolated strains from patients with PID were subjected to identification, serotyping and antimicrobial susceptibility testing. Results: From 796 strains analyzed, 14.8% ( n = 118) were resistant to penicillin, being 3% ( n = 24) with intermediate resistance and 11.8% ( n = 94) with full resistance, especially in patients with meningitis. Moreover, resistance to ceftriaxone was 5.3%: 34 (4.3%) with intermediate resistance and 8 (1%) with full resistance. We point out that the greatest level of resistance profiles was observed against sulfamethoxazole/trimethoprim (SMT): 350 (49.4%). On the other hand, antimicrobial susceptibility was described above 90% to chloramphenicol: 99.6% ( n = 696), erythromycin: 94.7% ( n = 664), ceftriaxone: 94.7% ( n = 754) and fully susceptible to vancomycin. Among the 18 most common serotypes, 9V and 14 showed less susceptibility to SMT, to penicillin and ceftriaxone; 19A to SMT and penicillin; 1 to SMT; 12F and 3 to chloramphenicol; 6A to SMT; 6B 23F to erythromycin and penicillin. Conclusion: Monitoring of Streptococcus pneumoniae antimicrobial resistance is essential to guide the appropriate empirical treatment of pneumococcal disease.


introDuCtion
Infections by Streptococcus pneumoniae persist as an important health problem worldwide (1) , despite the extensive investment in vaccines and the availability of appropriate antibiotic therapy.We must highlight that pneumococcus is part of human nasopharyngeal microbiota and can be isolated including in healthy adults and children who attend institutions of early childhood education.In Brazil, the prevalence of pneumococcus carriers ranges from 13% to 72%, depending on age and presence of associated diseases (2) .
In the decade of 1940s, the advent of penicillin apparently controlled pneumococcal diseases.However, as years went by, an increase in resistant strains was observed, both to classical and to the latest-generations treatments (3)(4)(5)(6)(7)(8)(9)(10) .In Brazil the first report of resistance to pneumococcus was delivered in 1988 (11) .
The indiscriminate use of antibiotic drugs is considered a risk factor for the increase in the resistance profile of S. pneumoniae, favoring colonization of nasopharynx and the occurrence of pneumococcal invasive diseases (PID) by resistant strains (1,6) .Researches about PID exhibit regional and seasonal specificities, what determines the need of periodic monitoring for the 10.5935/1676-2444.20170029establishment of interventions and control policies.Additionally, surveillance of the antimicrobial susceptibility pattern of strains is enormously valuable in the choice of the initial empirical treatment, as well as for the rational use of antimicrobials (12) .
This study was aimed at evaluating the susceptibility profile of different serotypes of S. pneumoniae isolated from individuals with PID in the northeastern macroregion of São Paulo state, Brazil, during a period of 16 years.

MEthoD
This is a retrospective cross-sectional research focusing secondary data on the antimicrobial susceptibility profile of S. pneumoniae isolated from patients with PID in a period of 16 years.Those patients were from the northeastern region of the state of São Paulo, Brazil, registered at the Regional Healthcare Network 13 (RRAS 13), which is part of the Regional Health Department (DRS) of Araraquara, Barretos, Franca, and Ribeirão Preto, with 90 municipalities (13) .
Information about serotypes and antimicrobial susceptibility profile were collected from 1998 to 2013 at the Center of Regional Laboratory (CLR) of Instituto Adolfo Lutz in Ribeirão Preto (IAL-RP) and Central IAL/São Paulo.It is appropriate to stress that the Bacteriology Center of IAL acts as a reference laboratory at state and national level for meningitis and pneumococcal infections.
Data were analyzed by the Statistical Package for the Social Sciences (SPSS), version 19.0, for the chi-square association test between distribution of cases according to period, clinical diagnosis, serotype, or susceptibility of each antimicrobial drug.
The project was approved by the Human Research Ethics Committee (report no.26759714.80000.5393).
A total of 69.2% of S. pneumoniae were susceptible to penicillin and ceftriaxone according to the oxacillin disk screening test.For 30.8% of oxacillin-resistant strains, the minimum inhibitory concentration (MIC) was determined.
For chloramphenicol erythromycin, and sulfamethoxazole/ trimethoprim (SMT), the disk-diffusion test defined the profile of susceptibility of S. pneumoniae.There was an increase in the pneumococcus susceptibility to SMT after vaccination in 2010 (Table 1).Among the non-susceptible S. pneumoniae (intermediate and resistant), rates of 53.5% for SMT, 14.8% for penicillin, 5.2% for erythromycin, and 0.4% for chloramphenicol occurred.A decrease was evident in susceptibility of pneumococcus to penicillin from 1998-2002 to 2003-2010.Susceptibility to ceftriaxone remained above 90%; however there was a progressive reduction of these rates during the studied period (Table 1).
Out of the 796 pneumococcus strains evaluated, 94 (11.8%) were totally resistant to penicillin, with 11.7% isolated from meningitis and just 0.1% from pneumonia.Only 3% of the S. pneumoniae isolated from non-meningeal infections presented intermediate resistance to penicillin (Table 3).
Seven cases (0.9%) of multi-resistant S. pneumoniae occurred (resistance to three classes of antimicrobials), with four being isolated from meningitis, two from pneumonia, and one from sepsis.In all of them, simultaneous resistance to penicillin, erythromycin, and SMT was observed, with 14, 19F, 6A, and 6B being the most involved serotypes.

DiSCuSSion
Monitoring antimicrobial resistance of S. pneumoniae is fundamental to guide the empirical treatment of PID, as well as to encourage reflections to support public immunization policies.
Lineages of penicillin-resistant S. pneumoniae are not limited to infectious pictures.In Brazil, there are reports of this profile in asymptomatic carriers, mainly in children attending day-care centers, a fact that can be associated with a greater use of antimicrobials (6) .In Portugal, evaluation of invasive and non-invasive respiratory pneumococcal infections in all age groups revealed that 18.4% of the strains were not susceptible to penicillin (19) , a percentage higher than the one found in the current research (14.8%).
Regarding S. pneumoniae susceptibility to chloramphenicol, erythromycin, and SMT, Santos et al. (2013) (20) , evaluating the periods before and after introduction of the 10-valent pneumococcal conjugate vaccine (PCV10), in São Paulo, found a similar result to that of the region in this study.In that research, S. pneumoniae were less resistant to erythromycin than those presented in Germany (19.9%) (8) and United States (35.3%) (21) .
In the present research pneumococcus demonstrated higher level of resistance to SMT, data compatible with the evidences of other national researches (12,20,22) .Soeters et al. (2011) (12) , in South Africa, showed evidence that non-susceptibility of pneumococcus to SMT is generally associated with resistance to multiple drugs, a fact observed in patients with the human immunodeficiency virus (HIV) that use SMT as a prophylactic drug for prevention of opportunistic infections.

The reduction of susceptibility of pneumococcus to penicillin
Penicillin resistance restricted to serotypes 14, 23F, 19F, 9V, 19A, 6B e 6A coincides with serotypes traditionally described as penicillin-resistant in other researches (4,7,22,24,25) .Serotypes 19A and 6A are not part of PCV10, available in public health care centers, but there is cross-reactivity between serotypes 6B/6A and 19F/19A (26) .These serotypes are related to nasopharyngeal colonization in children (27) , thus being more prone to develop reduced susceptibility to penicillin and to macrolides because these are antimicrobials routinely used in the community (21) .
In the period before the introduction of conjugated vaccines, the emergency and dissemination of international clones Spain 9V -ST156 and Tennessee 14 -ST67 increased resistance to antimicrobials (25) both here and in other Latin American countries (7) .In Brazil, those clones express the capsule of serotype 14 (25) .In Rio de Janeiro, Barroso et al. (2012) (4) isolated, from patients with meningitis, penicillin-resistant S. pneumoniae (clone Spain 9V -ST156) linked to serotype 14.Several authors reported rates of penicillin resistance of S. pneumoniae, isolated from patients with meningitis, ranging from 21.4% to 27.1% (3,7,22,28) , compatible with the 25.5% rate found here.On the other hand, lower values were observed in the state of Paraná (15%) (23) and in Salvador (13%-19%) (29) , and higher than 50% in Cuba, El Salvador, Honduras and Mexico (7) .
For the other non-meningeal pneumococcal infections, our research detected 3% of strains with intermediate resistance and 0.1% with full resistance to penicillin.According to Negrini (2010) (28) , in the municipality of Ribeirão Preto, children younger than 5 years presented 3.5% of S. pneumoniae with intermediate resistance, and none presented full resistance to penicillin.In 2012, in Brazil, full resistance was not observed to penicillin either, and intermediate resistance was 7.5% and 3.9% for children younger and older than 5 years, respectively.In Argentina, for children younger than 5 years, 100% of the strains were susceptible to penicillin (7) .
In this study, among the pneumococci isolated from patients with pneumonia, just 0.1% was totally resistant to penicillin, and 3% of the isolates from non-meningeal infections presented intermediate resistance to penicillin.This piece of data reinforces the consensus of some researches that concluded that patients with pneumonia treated with penicillin presented similar clinical evolution, regardless of whether the in vitro susceptibility test result was resistant or sensitive to penicillin (28) .Therefore, for nonmeningeal infections, penicillin continues to be the treatment of choice for pneumococcal diseases (30) .
In order to treat intermediate resistance infections, habitual doses of penicillin and derivatives are efficient in the treatment of community-acquired pneumonia.However, for the treatment of acute community-acquired bacterial meningitis, third-generation cephalosporin is the empirical initial treatment (23) .
With the introduction of conjugated pneumococcal vaccines, besides the expressive reduction in PID, the decline of infections by strains non-susceptible to penicillin was documented, due to decreased serotypes associated with resistance present in the composition of vaccines (1,7) .
In the city of Uberlândia (MG), 27.8% strains with diminished susceptibility to oxacillin were detected, a result similar to the 30.8% of this study.However, for ceftriaxone, the study in Uberlândia detected 12.5% resistance, a rate much higher than the one found in the current study (3) .In Guatemala and Mexico, a superior proportion of S. pneumoniae resistant to ceftriaxone (16.7% and 16.5%, respectively) (7) for non-meningeal infections was verified.
In many countries of Latin America, full resistance to ceftriaxone in pneumococcal meningitis isolates is low, with the highest percentage observed in Mexico (32.3%).In Brazil, in nonmeningeal infections, a rate of 3% of intermediate resistance to ceftriaxone was reported, with no isolation of the pneumococcus with full resistance (7) , a value similar to the one of the region researched in this work.
In general, the resistance profile of S. pneumoniae to ceftriaxone is low, yet, the importance of monitoring resistance levels is stressed due to the excellence of this antimicrobial for treatment of bacterial meningitis (29) .Strains of S. pneumoniae resistant to penicillin are frequently associated with resistance to other classes of antimicrobials, especially SMT (12) .In Porto Alegre, among the 18 penicillin-resistant pneumococcus strains, seven were resistant to at least two other drugs (9) .In this research, 0.9% of multi-resistant S. pneumoniae was reported, with simultaneous resistance to penicillin, erythromycin, and SMT, confirming the most frequent profile of S. pneumoniae with multi-resistance.The isolation of multiresistant lineages represents a world threat; in the USA it commonly relates to serotype 19A after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) (30) .Our results indicate that serotypes 14, 19F, 6A and 6B were involved in this multiresistance profile.Besides, S. pneumonia with full and simultaneous resistance to penicillin and ceftriaxone were observed in eight (1%) of the patients with meningitis, a value lower than the 13% detected in Rio de Janeiro in isolates of patients with meningitis (4) .
Additionally, the relationship of serotype 1 with PID and with colonization of nasopharynx just in short periods is stressed.It is less exposed to the selective antimicrobial pressure, and consequently, it has a lower level of resistance to penicillin (24) .

ConCLuSion
S. pneumoniae lineages presented susceptibility above 90% to chloramphenicol, erythromycin and ceftriaxone during the whole studied period, with total susceptibility to vancomycin.Nonsusceptibility to penicillin stands out, detected in 14.8% of the strains, with 11.8% with full resistance, mainly in patients with meningitis.Resistance to penicillin was restricted to serotypes 14, 23F, 19F, 9V, 19A, 6B and 6A.The highest level of resistance was detected for SMT.
Serotypes 9V, 14, 19A and 23F were less susceptible to penicillin than the other serotypes, while 7F, 1, 12F, 18C, 3, 4 and 5 were more susceptible to penicillin than the most frequent serotypes in the region.

taBLE 3 −
Main PID in patients at the RRAS 13, according to the profile of susceptibility to penicillin and ceftriaxone, 1998-2013 %*: refers to the total of strains tested for each antimicrobial; **: abscess, arthritis, peritonitis, pancreatitis, cirrhosis, surgical site infection, and appendicitis.PID: pneumococcal invasive disease; RRAS 13: Regional Healthcare Network 13.