Evolution of Antimicrobial Susceptibility to Penicillin in Invasive Strains of Streptococcus pneumoniae during 2007–2021 in Madrid, Spain

The use of pneumococcal conjugate vaccines has affected the epidemiology and distribution of Streptococcus pneumoniae serotypes causing Invasive Pneumococcal Disease (IPD). The aim of this study was to analyze the evolution of the phenotypical profiles of antimicrobial susceptibility to penicillin (PEN) in all IPD strains isolated in Madrid, Spain, during 2007–2021. In total, 7133 invasive clinical isolates were characterized between 2007 and 2021. Levels of PENR and PNSSDR were 2.0% and 24.2%, respectively. In addition, 94.4% of all the PENR belonged to four serotypes, including 11A (33.6%), 19A (30.8%), 14 (20.3%) and 9V (9.8%). All the strains of serotype 11A, which is a non-PCV13 serotype, were detected after the year 2011. Serotypes 6C, 15A, 23B, 24F, 35B, 19F, 16F, 6B, 23F, 24B, 24A, 15F and a limited number of strains of serogroups 16 and 24 (non-typed at serotype level) were associated with PNSSDR (p < 0.05). PNSSDR strains of non-PCV13 serotypes 11A, 24F, 23B, 24B, 23A and 16F were more frequent from 2014 to 2021. The changes in S. pneumoniae serotype distribution associated with the use of conjugate vaccines had caused in our region the emergence of non-PCV13 pneumococcal strains with different PENR or PNSSDR patterns. The emergence of serotype 11A resistant to penicillin as the most important non-PCV13 serotype is a worrisome event with marked relevance from the clinical and epidemiological perspective.


Introduction
Vaccination with the 23-valent pneumococcal polysaccharide vaccine (PPV23) was recommended in Spain in 2001 for individuals aged more than two years old who were at high risk of pneumococcal disease [1]. In 2005, some regions, such as the Autonomous Community of Madrid, extended its use to adults over 59 years old. In addition to PPV23, pneumococcal conjugate vaccines (PCVs) have been included in the Madrid region [2][3][4]. First, the 7-valent pneumococcal conjugate vaccine (PCV7) that included serotypes 4, 6B, 9V, 14, 18C, 19F and 23F was introduced in 2001 in the private paediatric market. In November of 2006, Madrid included this vaccine in the children's vaccination program [2]. The use of PCV7 affected the distribution of serotypes in the Spanish population, with changes in the penicillin susceptibility patterns of Streptococcus pneumoniae [5,6]. Thus, the non-susceptibility to penicillin level decreased from more than a half at the beginning of this century to near one third at the end of the first decade [6]. After the use of the PCV7, the rise in the penicillin non-susceptible serotype 19A (not covered by this vaccine) became very prevalent [6]. In 2010, PCV7 was replaced by the 13-valent pneumococcal conjugate vaccine (PCV13), containing serotypes covered by PCV7 plus 1, 3, 5, 6A, 7F and 19A; it was introduced for the paediatric population. In 2012, PCV13 was removed from the funded immunization program in Madrid with private administration according to the individual recommendations of paediatricians and this vaccine was finally implemented in the Spanish national immunization childhood calendar in 2016 [2][3][4]. Its use in children has affected the epidemiology of the invasive pneumococcal disease (IPD), reducing the incidence in children but also in adults due to its herd protection effect. The decrease in PCV13 serotypes since 2010 was accompanied by a decrease in penicillin non-susceptibility within PCV13 isolates [5]. This reduction was mainly due to the fall of the serotype 19A incidence [7]. However, the reduction in covered PCV13 serotypes after the use of this vaccine was followed by the emergence of non-PCV13 serotypes [4,5,8,9]. Antimicrobial resistance has been proposed as one of the top ten public health threats by the World Health Organization. To reinforce the discovery of new antibiotics, artificial intelligence strategies have been proposed to identify new antibiotics and even to predict the evolution of vaccine preventable diseases [10][11][12]. In this context of high rates of antibiotic resistance and serotype replacement by non-vaccine serotypes, the aim of this study was to analyze the evolution of the phenotypical profiles of antimicrobial susceptibility to penicillin in IPD isolates from Madrid, Spain, during the period 2007-2021.

Discussion
Microbiological surveillance studies deciphering circulating serotypes and resistance profiles are critical for understanding local epidemiology of IPD, for assessing the impact of current and future vaccines and for monitoring antimicrobial susceptibility. However, different breakpoints to penicillin in S. pneumoniae depending on the guidelines (EUCAST or CLSI), variations in national immunization calendars by country and the implementation of different surveillance systems make the comparison of results difficult [13,14]. In the year 2008, CLSI changed the penicillin cut-off that was largely used until 2007 [15,16]. In 2020, EUCAST introduced a change in the intermediate criteria considering the strains in this category as susceptible with increased exposure, assuming that there is a high probability of therapeutic success by increasing the antimicrobial concentration [17]. This change has been maintained in the 11th EUCAST 2021 version [18]. The main reason for this recommendation is to detect treatable infection rather than the identification of resistance mechanisms [14]. The use of the old definition of "intermediate" crafted by EU-CAST 2002-2018 [19] had proved to be difficult in clinical practice and EUCAST now categorises as "susceptible increased exposure" when there is a high likelihood of therapeutic success, because exposure to the agent is increased by adjusting the dosing regimen or by its concentration at the site of infection. However, previously, surveillance systems considered the categories intermediate and resistant under a wide definition of non-susceptibility [20]. In this study, in order to maintain traceability with the scientific literature, it has been chosen to consider the categories of PENR and PNSSDR (this last corresponded to the term "non-susceptible" usually applied). In other studies using the same antimicrobial methodology, resistant and intermediate isolates were all referred to as non-susceptible [21,22]. Although E-test has been widely used to perform antimicrobial susceptibility testing of S. pneumoniae, this method is not considered by CLSI or EUCAST in their guidelines, and, therefore, it may represent a limitation of the current study.
In terms of pneumococcal epidemiology and the contribution of vaccines to ameliorate the antimicrobial resistance problem, due to its herd protection, the use of pneumococcal conjugate vaccines has led to a decrease in the incidence of vaccine serotypes associated with the decline in antibiotic resistance [23,24]. However, after the introduction of the pneumococcal conjugate vaccines (PCV7 and, later, PCV13), a rise in non-vaccine serotypes displaying antibiotic-resistant has been identified [24,25]). A clear example of this phenomenon is represented by serotype 19A (included in PCV13 but not in PCV7). This was one of the most prevalent PENR serotypes after the use of PCV7 [26]. The variation in the incidence and antibiotic resistance of this serotype has been very evident with the

Discussion
Microbiological surveillance studies deciphering circulating serotypes and resistance profiles are critical for understanding local epidemiology of IPD, for assessing the impact of current and future vaccines and for monitoring antimicrobial susceptibility. However, different breakpoints to penicillin in S. pneumoniae depending on the guidelines (EUCAST or CLSI), variations in national immunization calendars by country and the implementation of different surveillance systems make the comparison of results difficult [13,14]. In the year 2008, CLSI changed the penicillin cut-off that was largely used until 2007 [15,16]. In 2020, EUCAST introduced a change in the intermediate criteria considering the strains in this category as susceptible with increased exposure, assuming that there is a high probability of therapeutic success by increasing the antimicrobial concentration [17]. This change has been maintained in the 11th EUCAST 2021 version [18]. The main reason for this recommendation is to detect treatable infection rather than the identification of resistance mechanisms [14]. The use of the old definition of "intermediate" crafted by EUCAST 2002-2018 [19] had proved to be difficult in clinical practice and EUCAST now categorises as "susceptible increased exposure" when there is a high likelihood of therapeutic success, because exposure to the agent is increased by adjusting the dosing regimen or by its concentration at the site of infection. However, previously, surveillance systems considered the categories intermediate and resistant under a wide definition of non-susceptibility [20]. In this study, in order to maintain traceability with the scientific literature, it has been chosen to consider the categories of PENR and PNSSDR (this last corresponded to the term "nonsusceptible" usually applied). In other studies using the same antimicrobial methodology, resistant and intermediate isolates were all referred to as non-susceptible [21,22]. Although E-test has been widely used to perform antimicrobial susceptibility testing of S. pneumoniae, this method is not considered by CLSI or EUCAST in their guidelines, and, therefore, it may represent a limitation of the current study.
In terms of pneumococcal epidemiology and the contribution of vaccines to ameliorate the antimicrobial resistance problem, due to its herd protection, the use of pneumococcal conjugate vaccines has led to a decrease in the incidence of vaccine serotypes associated with the decline in antibiotic resistance [23,24]. However, after the introduction of the pneumococcal conjugate vaccines (PCV7 and, later, PCV13), a rise in non-vaccine serotypes displaying antibiotic-resistant has been identified [24,25]). A clear example of this phenomenon is represented by serotype 19A (included in PCV13 but not in PCV7). This was one of the most prevalent PENR serotypes after the use of PCV7 [26]. The variation in the incidence and antibiotic resistance of this serotype has been very evident with the introduction of each vaccine in the childhood immunization schedule [4,26,27]. This pattern is clearly reflected in our study, performed using clinical isolates from Madrid, confirming a decrease in the number of IPD cases of serotype 19A and the association of antibiotic resistance after the introduction of PCV13.
Epidemiological analysis confirmed that changes in the distribution of S. pneumoniae serotypes associated with the use of conjugate vaccines had caused in our region the selection of strains belonging to other serotypes with different PENR or PNSSDR patterns. Others authors have noted that susceptibility patterns are serotype-specific [28]. In any case, mutations in the genes coding the penicillin-binding proteins (PBPs) have been recognized in S. pneumoniae as the major resistance mechanism for β-lactam antibiotics [29], indicating that the emergence of resistant strains can be spread by clonal propagation [30,31]. Thus, the main limitation of the present study is that the analysis was performed at the phenotype level with no information about the molecular resistance mechanisms or the genotypes involved.
In addition, the association of some serotypes such as 11A, 14, 24F and 23B with non-susceptibility to penicillin has been previously described [5,25,32]. In this sense, the increase in beta-lactam resistance among serotype 11A was linked to a clonal shift in this serotype [23,25,33]. In Spain, serotype 11A strains isolated in 2010-2011 already showed a penicillin MIC90 coinciding with the resistant EUCAST breakpoint of 2mg/L [6]. This serotype is currently among the most prevalent causing IPD in our country [4]. Thus, the emergence of penicillin-resistant strains of serotype 11A is concerning from a pathogenesis perspective [34]. The invasive disease potential of this serotype is highly related to the rise of genotype ST6521 11A that has spread across Europe in the last years, becoming one of the most prevalent within serotype 11A [35]. This genotype of serotype 11A is associated with high levels of antibiotic resistance, shows a greater ability to form biofilms and avoids very efficiently the host immune response [34]. In a recent study conducted by our group, this prevalent serotype was detected with the second highest fatality rate [36] and has shown an increase in penicillin resistance in the last years. Indeed, the pandemic of SARS-CoV-2 has increased the resistance levels of circulating strains of serotype 11A in Spain, with an increase in MIC90 to penicillin from 2mg/L during 2016-2019 to 4mg/L in 2020 [26]. Another alarming non-PCV13 serotype that has emerged within the PNSSDR strains is 24F. This serotype displays resistance to penicillin and its prevalence in the paediatric and adult population is increasing in some countries, including Spain [4,26].
In the last years, there has been a decrease in the incidence of many of the resistant strains but also in the incidence of IPD due to the COVID-19 pandemic [37]. The surveillance of the behaviour of serotypes and their resistance to antibiotics will be crucial for the development of new vaccines and the implementation of vaccination schedules that can help to prevent IPD. The use of vaccines in national immunization schedules is a costeffective measure to decrease antibiotic resistance [38,39], and, probably, the inclusion of serotypes 11A and 24F in future vaccines for more of the population could be crucial for preventing penicillin resistance and non-susceptibility [40,41].

Materials and Methods
Invasive pneumococcal strains from IPD cases (one for every episode) isolated from 2007 to 2021 were sent from the Microbiological Laboratory Services of Public and Private Hospitals located in the Madrid Region (Spain) to the Madrid Public Health Regional Laboratory for serotyping and antimicrobial susceptibility. In this study, 7,133 clinical isolates from IPD were characterized. Identification of the capsular serotypes was carried out by the Pneumotest-Latex (Statens Serum Institut, Copenhagen, Denmark) and by Quellung reaction using commercial antisera (Statens Serum Institut, Copenhagen, Denmark).
To perform the antimicrobial susceptibility testing of S. pneumoniae by the E-test method, commercial strips (Benzylpenicillin ETEST ® strips; bioMérieux España S.A) with a concentration rank of 0.002-32mg/L were used. The inoculum was adjusted to a bacterial concentration of 0.5 McFarland standard (or 1 McFarland standard if mucoid strain) and the S. pneumoniae ATCC 49619 was employed as the reference strain. The strips were applied to the surface of inoculated Mueller-Hinton, supplemented with 5% of sheep blood. Agar plates were incubated at 35 ± 2 • C in a 5%CO2 atmosphere for 20 to 24h. MIC values were obtained from the scale at the intersection point between the complete inhibition ellipse edge and the strip. According to the CLSI breakpoints (PEN non-susceptibility for the oral treatment of non-meningitis syndromes) [42] and the EUCAST breakpoints (Table  S1) [17,18], pneumococcal strains showing MIC > 2mg/L and >0.06mg/L were categorized, respectively, as PEN resistant (PENR) and PEN non-susceptible at standard dosing regimen (PNSSDR). S. pneumoniae strains with MICs 0.094-0.5mg/L were considered PNSSDR of a low level. Pneumococcal isolates with MIC > 0.5-2mg/L were considered PNSSDR of a high level. To analyze the evolution of serotypes causing IPD and the pattern of penicillin susceptibility during the period 2007 to 2021, the Odds Ratio (OR) with its correspondent 95% confidence intervals (CI95) were calculated. The statistical significance was set at p < 0.05. Statistical analyses were performed using STATA v16. In order to relate the use of the conjugate vaccines across time, the entire temporal series was divided into the late PCV7