Streptococcus pneumoniae remains one of the leading community-acquired bacterial pathogens worldwide. The spectrum of pneumococcal diseases ranges from non-invasive, such as acute otitis media (AOM), sinusitis, and non-bacteremic pneumonia, to invasive as bacteremic pneumonia, sepsis, and meningitis. Young children and the elderly are the most affected age groups, with more than 300,000 deaths annually attributable to S. pneumoniae in children younger than five years (Wahl et al., 2018) and more than 690,000 deaths in adults over 70 years in 2015 worldwide (Troeger et al., 2017). However, invasive pneumococcal disease (IPD) dramatically declined in children and adults following the introduction of pneumococcal conjugate vaccines (PCVs) (CDC, 2019). Due to the phenomenon of serotype replacement and the emergence of non-vaccine serotypes, higher-valent PCVs (PCV10, PCV13, PCV15, and PCV20) replaced first-generation vaccines (PCV7). By the end of 2020, 148 out of 194 WHO member states have introduced PCV into their National Immunization Programs (NIPs) (CDC, 2020). Although the PCV7 was licensed in Serbia in 2009, and PCV10 and PCV13 in 2013, they have been available on a voluntary basis, and the coverage rate of PCVs was very low up to 2018. Thereafter, PCV10 began to be used routinely for immunization of children under 2 years of age in 2018,¹ and in 2022 it was replaced by PCV13. PCV10 vaccine coverage in 2018 did not exceed 50%.² In the period 2018-2021, vaccine efficacy and effectiveness and the potential serotypes shift could not be well monitored due to the COVID-19 pandemic and subsequently reduced number of IPD cases and low quality of IPD monitoring. The pneumococcal polysaccharide vaccine (PPV23) has been available in Serbia for over 20 years for risk groups.

Reliable epidemiological surveillance of infectious diseases is critical for making rational decisions on public health issues such as vaccination strategies. The choice of PCV to be used in a country should be primarily based on the local and regional prevalence of vaccine serotypes, antimicrobial resistance patterns, vaccine availability, vaccine cost, etc. (World Health Organization, 2021). In Serbia, IPD is notifiable according to the current Law on Protection of the Population from Infectious Diseases.³ Despite the legislative obligation, in practice, it is not fully implemented, and data on the incidence of IPD in our country is scarce. So far, no population-based study on IPD has been performed in Serbia. However, National Reference Laboratory (NRL) for Streptococci performs voluntary-based laboratory surveillance on IPD and serotype distribution (Gajic et al., 2013; Delic et al., 2021).

Antibiotic treatment of pneumococcal infections typically includes beta-lactams and macrolides, in addition to fluoroquinolones in adults. Due to the high antibiotic consumption in Serbia (Tomas et al., 2021) as well as very common non-invasive pneumococcal diseases that are treated with antibiotics, the rate of macrolide resistance among the invasive pneumococci in Serbia in 2020 was 31.8%, while up to 48.1% of isolates expressed decreased susceptibility to penicillin (WHO Regional Office for Europe/European Centre for Disease Prevention and Control, 2022). It is known that clonal expansion is considered to be the main mechanism for ongoing antibiotic resistance (Keenan et al., 2015). Also, the pneumococcal population structure is highly diverse, varies between geographic locations, and evolves and adapts to the selective pressure of PCVs (Lo et al., 2019). Therefore, information on the genetic background in circulating S. pneumoniae isolates will allow us to analyze further changes as a result of the implementation of PCV in Serbia.

Continuous surveillance of IPD is important both prior to and after vaccine introduction. Serotype distribution in the pre-vaccine period provides a baseline for evaluating the impact of PCV on S. pneumoniae epidemiology. Therefore, this study aimed to characterize invasive isolates of S. pneumoniae obtained up to the introduction of the PCVs in the Serbian NIP by determining: serotype distribution, the prevalence and genetic basis of antimicrobial resistance, and the genetic relatedness of the circulating pneumococcal clones.

This is the first comprehensive nationwide study on the characterization of circulating clones of invasive S. pneumoniae isolates in Serbia. Data from this study provide a baseline for future studies monitoring the impact of PCVs on serotype distribution and provide recommendations for necessary changes in the immunization schedule. They also shed light on the coverage of PPV23, a vaccine mandatory for certain patient categories since 2020.⁷

According to the NRL-based surveillance, the annual notification rate of IPD varies between 0.97 and 1.14 cases per 100,000 population in the last three years of the study. Observed rate is much lower than in the Netherlands (16), Sweden (13.9), Slovenia (13.4), and Norway (11.0) but higher than in Bulgaria (0.3), Romania, and Greece (each 0.4) and Croatia (0.5) in 2018 (ECDC, 2018). There are several reasons why IPD incidence is certainly underestimated in Serbia: primarily due to the voluntary-based reporting of IPD, insufficient blood sampling for blood culture, starting antibiotic therapy before sample collection, suboptimal standard culture techniques, voluntary sending of bacterial isolates, etc. Consequently, NRL receives a relatively small number of invasive pneumococcal isolates per year. Therefore, there is a clear need for improvement of blood specimen collection practices and IPD surveillance in our country because, despite the legislative obligation, it is poorly implemented. Also, the introduction of molecular methods in the routine practice would certainly improve diagnosis and surveillance of IPD. Since the detection of the lytA gene is not absolutely specific to S. pneumoniae (Tavares et al., 2019), the combination of target genes (psaA, wzg/cpsA, etc.) has been suggested to improve the reliability of the identification.

IPD was predominantly reported in adults (69.2%), particularly in persons between 18 and 65 years old (41.8%) and children ≤2 years old (20.6%). Obtained results are not in line with the report of the European Centre for Disease Prevention and Control (ECDC) for 2018 (ECDC, 2018), where the highest incidence of IPD was among patients ≥65 years. However, it is in accordance with the findings of Marrie T et al., who reported that 27.3 and 53.5% of the IPD cases were in persons aged ≥65 and 17-54 years, respectively (Marrie et al., 2018). On the other hand, the percentage of isolates in infants (20.6%) and the elderly ≥65 years (27.4%) correlates well with ECDC data (ECDC, 2018). Blood was the most common specimen source (59%) obtained from cases of septicemia/occult bacteriemia and bacteremic pneumonia, followed by CSF (34.3%) taken from meningitis cases and pleural fluid (6.1%) collected from patients with pleural empyema. ECDC reported slightly different results, indicating that the most common clinical presentations of IPD in European countries were septicemia (35%), bacteremic pneumonia (43%), and meningitis (19%) (ECDC, 2018). The discrepancy in the incidences of meningitis cases between our and EU data may be due to the fact that clinical presentation was known for 34% of all IPD cases in the EU and that PCVs have been introduced in EU countries before than Serbia, resulting in the lower incidence of IPD in infants and decline in meningitis cases.

In the present study, almost two-thirds of all pneumococcal isolates belonged to the most common seven serotypes (3, 19F, 14, 6B, 6A, 19A, and 23F), which corresponds to the pre-PCV period in most European countries, such as Finland (14, 3, and 23F), Bulgaria (3 and 19F), Slovenia (14, 9 V, 3, 7F, 19A, and 1) and the United States (6B, 14, 19F, 23F, 3, 6A, and 19A) (Setchanova et al., 2012; Richter et al., 2013; Jokinen et al., 2015; Müller Premru et al., 2015).

Over the nine-year study period, the coverage rate of PCV10 among children <5 years declined from 84.6% in 2010 to 53.3% in 2018, while the coverage of PCV13 slightly decreased from 84.6% in 2010 to 73.3% in 2018. Also, the coverage rate of PPV23 in those older than 2 years was quite stable (84.1% in 2010 vs. 80.8% in 2018). Immediately after the introduction of vaccination, in the second decade of the 2000s, the coverage rate of PCV10 and PCV13 on all IPD cases in Austria, Netherlands, and Bulgaria were 71 and 93%, 42, and 60%, 59.9 and 78.8% (Setchanova et al., 2012; Netherlands Reference Laboratory for Bacterial Meningitis (AMC/RIVM), 2014; Paulke-Korinek et al., 2014).

Unlike in most countries, a high incidence of serotype 3 was registered in the pre-vaccine period in Serbia. Reports from countries that have used PCV10 indicated a remarkable increase in PCV13 non-PCV10 serotypes, particularly 3 and 19A. Although some studies (Domingues et al., 2014; Deceuninck et al., 2015; Jokinen et al., 2015) suggest that PCV10 may provide cross-protection against 19A IPD in young children, no reduction in serotype 19A-related IPD was seen in older children and in adults. In fact, serotype 19A became significantly and increasingly prevalent, particularly in PCV10 countries. Thus, in Austria, serotype 19A increased from 3% of all IPD cases in the pre-PCV to 6–7% in just 2 years (Paulke-Korinek et al., 2014). Also, in Finland, 4 years after PCV10 introduction, the most prevalent serotypes were 19A (30%) and 3 (19%) (Jokinen et al., 2015). Further studies should evaluate the potential increase in these serotypes in Serbia.

It should be noted that IPD coverage of PPV23 in our adult population is high and exceed 85%, as it was provided that this vaccine could provide moderate long-term protection against hospitalization with PPV23 serotype pneumonia and IPD (Lawrence et al., 2020).

As the consumption of antibiotics in Serbia is still very high, especially broad-spectrum macrolides, quinolones, and third-generation cephalosporins (Tomas et al., 2021), bacterial resistance is on the rise. Results on antimicrobial resistance of invasive S. pneumoniae are in line with previous findings that reported high levels of pneumococcal non-susceptibility to penicillin (47.6%) and macrolides (40.4%) in Serbia (Gajic et al., 2013; Delic et al., 2021). Particularly worrying is the non-susceptibility to the third-generation cephalosporins (16.6%) and high levels of MDR (20%) and XDR (16.9%) isolates, which mainly express simultaneous non-susceptibility to first-line anti-pneumococcal antibiotics. Observed macrolide non-susceptibility was 40.4%, mainly due to the cMLSB-phenotype (68.7%), which is a similar rate as in a number of European countries, including Belgium, France, and Spain in the pre-vaccine era, with an average rate of 62.3% (Farrell et al., 2008). In contrast, the M-phenotype associated with the mef genes was the predominant resistance mechanism in some European countries (Austria, Bulgaria, Ireland, Finland, Germany, Greece, United Kingdom) and the United States in the pre-PCV period (Farrell et al., 2008; Siira et al., 2009; Bley et al., 2011; Setchanova et al., 2012). In the present study, the simultaneous presence of both mef and erm genes was detected in 16.6% of the isolates, which is higher than the mean European value (6.1%) at that time (Berbel et al., 2022). However, 5% of the tested macrolide-resistant isolates were negative for both genes, indicating the possibility of other resistance mechanisms such as L4 and L22 ribosomal protein mutations, or the presence of other mef and erm gene variants (Nagai et al., 2002; Midouni Ayadi et al., 2020), but additional research is warranted. The rates of pneumococcal non-susceptibility to penicillin and macrolides in Serbia were generally higher than in most European countries in the period 2016-2018, such as Austria, Czech Republic, Netherlands, Germany, United Kingdom, France, Spain, Poland, and Italy (WHO Regional Office for Europe/European Centre for Disease Prevention and Control, 2022). However, it is comparable with those found in Belarus, Bosnia and Herzegovina, Cyprus, France, Iceland, Malta, Romania, and Turkey, where the non-susceptibility rates to penicillin and macrolides were above 25% (WHO Regional Office for Europe/European Centre for Disease Prevention and Control, 2022).

In the current study, the most resistant serotypes were 14, 19F, 6B, 6A, and 23F, which is comparable with results reported in other European countries and the USA before PCVs introduction (Dagan and Klugman, 2008). Serotype 19A has been typically associated with antibiotic resistance (Desmet et al., 2022); in this study, we did not find significant resistance in our 19A isolates. However, due to the high coverage of PCV10 and PCV13 of resistant S. pneumoniae isolates (74.6 and 85.1%, respectively), a decline in pneumococcal resistance in our country could be expected.

This study revealed circulating CCs of invasive pneumococcus in Serbia for the first time. The majority of the detected STs in Serbia have also been reported in the pre-PCV period worldwide (ST62, ST81, ST180, ST199, ST271, ST320) and in neighboring countries (ST15, ST66, ST156, ST191, ST230, ST473, ST1377) (Müller Premru et al., 2015; Li et al., 2019; Sheppard et al., 2019).⁸ In the present study, 44.3% of the detected clones were closely related to 11 of the 43 PMEN clones (Spain23F-1, Spain9V-3, Taiwan19F-14, Taiwan23F-15, Greece6B-22, Sweden15A-25, Netherlands3-31, Denmark14-32, Netherlands18C-36, Netherlands15B-37, and Netherlands7F-39). ST1377 was the most common genotype among serotype 3 isolates, while globally disseminated Netherlands3-31 (ST180) only represented 25% of our isolates.

As observed in some countries before the introduction or widespread use of PCV7, the MDR 19A serotype pneumococci were also identified in our population (Choi et al., 2008). The S. pneumoniae 19A isolates were genetically heterogenous and were assigned to eight different STs (ST179, ST199, ST416, ST645, ST878, ST2345, ST10432, and ST11340). However, the globally disseminated MDR CC320/271 genotype responsible for the post-conjugate vaccine increase in MDR serotype 19A (Ruiz García et al., 2021) represented 74.2% of our serotype 19F isolates, all of which were MDR. Similar findings were reported by Ndlangisa et al., where no 19A serotype isolates were detected among CC320/271 (Ndlangisa et al., 2014).

While exceptions exist, it is generally observed that isolates sharing the same ST or CC tend to exhibit the same serotype (Brueggemann et al., 2003). Therefore, a new or unusual serotype-sequence type combination could indicate capsular switching. Among our isolates, we identified new ST-serotype combinations. Based on the PubMLST database, ST230 is associated with multiple serotypes, but it has not been reported among serotype 7F isolates; however, one of our serotype 7F isolates was ST230. Similarly, among CC320/271 isolates commonly associated with serotypes 19A and 19F, one isolate with 6B serotype was detected.

This study has several limitations. Firstly, the isolates collected in this study were sent on a voluntary basis, and the number of isolates causing IPD is probably underestimated. Secondly, only randomly selected isolates were genotyped by MLST due to financial limitations.

Overall, our study provides a comprehensive insight into the epidemiological diversity of S. pneumoniae isolates causing IPD in Serbia before PCV introduction into NIP. Serotype 3 is dominant in adults, while 19F and 14 were the most common in children. The PCV10 and PCV13 may provide significant protection to our children ≤2 years, with 71.3 and 86.1% coverage, respectively. Our isolates are highly non-susceptibile to penicillin and macrolides, with worrisome rates of MDR and XDR strains. Substantial coverage of implemented PCVs should provide protection against invasive diseases and MDR strains. Also, there is considerable diversity among the isolates, with 60 circulating STs detected. Therefore, the distribution of serotypes, antimicrobial resistance, and clonal association of invasive pneumococcal strains as a response to PCV implementation should be further monitored.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

NO and IG contributed to the conception and design of the study. DK, MJ, ZV, DM, TT, SL, and JK were involved in the laboratory work and investigation. MJ and JK organized the database, prepared the figures and tables, and conducted the statistical analysis. NO, MJ, and JK wrote the sections of the manuscript. LR coordinated the study. All authors have read and agreed to the published version of the manuscript.

This work was supported partly by a research grant from Investigator-Initiated Studies Program of Merck, Sharp & Dohme doo (grant number 60165) and by the Ministry of Education, Science, and Technological Development of the Republic of Serbia, Serbia [grant No. 451-03-68/2022-14/200110]. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck, Sharp & Dohme doo.

NO acts as a principal investigator for investigator-initiated sponsored research related to the topic conducted on behalf of the Faculty of Medicine, University of Belgrade, for which the Faculty obtained a research grant from Merck Sharp and Dohme doo. There are no patents, products in development, or marketed products associated with this research. We fully adhere to Frontiers policies on data and material sharing.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.