Resistant Streptococcus pneumoniae strains in children with acute otitis media– high risk of persistent colonization after treatment

Streptococcus pneumoniae is both a commensal bacterium colonizing the human nasopharynx asymptomatically and a pathogen responsible for a wide spectrum of diseases, ranging from mild respiratory tract infections to severe invasive diseases. It is a major cause of morbidity in children and can be isolated in 25–60% of nasopharyngeal samples obtained from healthy children. Nasopharyngeal colonization can lead to infection, by spreading to adjacent mucosal tissue to cause acute otitis media (AOM) or pneumonia, or by blood stream to other sites causing bacteriemia, meningitis or focal infections [1]. The worldwide increase in antibiotic resistance in S. pneumoniae has been related to the spread of several pneumococcal serotypes (6A, 6B, 9 V, 14, 15A, 19F, 19A, and 23F), the so-called ‘paediatric serotypes’ which mainly belong to a small number of pneumococcal clones which nomenclature is standardized by the Pneumococcal Molecular Epidemiology Network (PMEN) [2–5].

Considering advances in the development of pneumococcal conjugate vaccines (PCVs) leading to reduction of invasive disorders, pneumococcal diseases are still problem for public health. The impact of the first introduced pneumococcal conjugate vaccine (PCV7) containing seven capsular antigens of serotypes 4, 6B, 9 V, 14, 18C, 19F and 23F on the decrease of invasive pneumococcal disease has been significant in infants, older children and adults. A decrease in rates of antimicrobial resistance among pneumococcal isolates was observed as an additional benefit of the vaccine because resistance to penicillin, macrolides and multidrug resistance are mostly associated with serotypes included in PCV7, namely 6B, 9 V, 14, 19F and 23F [6]. In 2007, World Health Organization (WHO) recommended to introduce PCV to the national infant immunization programs of all countries. Lower proportion of pneumococcal infections caused by PCV7 serotypes has been noted only in countries with routine effective use of PCV7 [7].

Acute otitis media (AOM) is a common childhood infection, occurring most frequently as a consequence of viral upper respiratory tract infections, but treated mainly with antibiotics. The leading causes of bacterial AOM worldwide are Streptococcus pneumoniae, non-typeable Haemophilus influenzae, Moraxella catarrhalis, and group A Streptococcus [8–10]. In approximately 80% of children aged 2–5 years AOM is diagnosed at least once, and 30–40% of them have recurrent episodes [8, 11]. More than 10% of children do not improve despite antibiotic therapy. It was noted that history of previous recurrent episodes of AOM, selective pressure of previous antibiotic courses and age below 2 years are risk factors related to this entity [12]. S. pneumoniae was shown to be the most prevalent pathogen among AOM patients who had failed a course of antibiotic therapy [12, 13]. It was demonstrated that the persistence of pneumococcal nasopharyngeal carriage is a risk factor for subsequent recurrent infections and the antimicrobial selection should have an impact on nasopharyngeal colonization [14, 15]. The nasopharynx is often a reservoir of bacteria involved in AOM where they interact with each other and the host’s immune system [16]. Disturbance of existing balance in this microbiota may facilitate the colonizing bacteria to expand through the Eustachian tube into the middle ear and cause infection [17].

In our previous study, we observed the high prevalence of antibiotic-resistant otopathogens in recalcitrant AOM and the high colonization rate by the same otopathogen species after completion of antibiotic therapy [10]. In this study we evaluate the relationship between the pneumococcal strains obtained from the nasopharynx (NP) and oropharynx (OP) and the middle ear fluid (MEF) of children during AOM and during post-treatment visits, based on their phenotypic and genotypic characteristics performed by analyses of serotype, antibiotic susceptibility patterns and multilocus sequence typing (MLST).

In this study of pneumococcal aetiology in children with different cases of AOM, high frequency of strains resistant to various antibiotic drugs was found. In fact, among all, 73.3% of isolated pneumococcal strains were obtained from patients with treatment failure and recurrent AOM. Antibiotics are prescribed at up to 80% of GP or ENT visits for AOM whereas antibiotic treatment is considered to be a selection factor of initially non-susceptible strains, facilitating colonization with new strains or development of resistance during therapy. S. pneumoniae was most prevalent pathogen isolated from MEF in patients who had failed a course of antibiotic therapy [13, 21]. Pichichero et al. [22] reported that 28% of AOM cases were caused by S. pneumoniae with high proportion of highly resistant strains and in 77% of cases isolation was done from recurrent AOM or initial treatment failure. Eldan et al. [23] found a significantly higher carriage rate of drug-resistant S. pneumoniae in patients with nonresponsive AOM than in children with simple, uncomplicated AOM who did not receive previous antibiotic therapy. In our study, 91.7% strains isolated from nasopharynx of patients with treatment failure and recurrent AOM were MDR ones. Complete concordance between resistance patterns and STs of pneumococcal isolates recovered from NP/OP and those isolated from MEF has proved that nasopharyngeal MDR strains were the etiologic agents of AOM in 70.6% (12/17) of patients with pneumococcal aetiology. Moreover, all of the pneumococcal isolates recovered from NP/OP during post-treatment visits after treatment were identical according to resistant patterns and STs with those isolated during AOM from the same patients. It is necessary to use molecular typing methods like MLST to find the genetic relatedness between bacterial isolates originating from middle ear cavity and naso/oropharynx. One study revealed with MLST analysis that 100% of pair pneumococcal isolates from nasopharyngeal and MEF samples were identical [24]. Using other genetic methods, it was observed that 71.4–80% of S. pneumoniae isolated from paired nasopharynx and MEF showed genetic identity [25, 26]. Our study and previous findings emphasize the hypothesis that nasopharynx acts as a reservoir for bacterial pathogens in AOM, including S. pneumoniae.

Tympanocentesis is not routinely performed procedure in Polish children which was the reason of relatively small number of enrolled patients. It is a limitation of this study. However, the results seem to confirm the relationship between the persistent pneumococcal colonization after unsuccessful eradication and presumably recurrence of an infection. It is worth noting that in 5 patients, whose MEF samples were S. pneumoniae positive during AOM, pneumococcal eradication was observed during post-treatment visit.

Some studies suggest that signs and symptoms of AOM caused by S. pneumoniae may be more severe (fever, severe earache, bulging TM) than those caused by other pathogens [27–29]. As well as it was observed elsewhere [30], in our study, higher values of CRP and WBC, whose levels rise in response to inflammation, were significantly important factors involved presence of S. pneumoniae in MEF sample in children with AOM.

A high level of penicillin-resistant S. pneumoniae is considered today the leading cause of antibiotic therapeutic failures in AOM [13–15, 23]. High frequency of PNSSP isolations in children with recalcitrant AOM, observed in our study, was also reported by other authors in other countries, including Poland [13, 23, 31, 32]. Furthermore, we noticed the difference between frequency of PNSSP isolations during AOM (86.4%, 19/22 strains) and during post-treatment visits (100%, 12/12 strains) caused by the selection pressure of used antibiotics. Moreover, in case of three strains isolated during post-treatment visits the increase of penicillin and cefotaxime MIC values were observed in comparison to isolates obtained during AOM.

Very high frequency (84%) of MDR strains in our studies has exceeded the data presented in other studies on AOM treatment failure, that 40–67% of pneumococcal strains were MDR [32, 33]. Our study have confirmed recent reports showing that antibiotic therapy in AOM induces selection of preexisting nasopharyngeal antibiotic-resistant S. pneumoniae [14, 34]. In our study, it was shown that too short time of therapy in children with bilateral AOM, but not of the type of β-lactams has the impact on persistent pneumococcal colonization after treatment.

In this study, the frequency of S pneumoniae isolation from MEF samples was 27.4% and it constituted 43.6% of positive MEF samples. There are similarities between our study and other studies using conventional culture method which reported relatively high rates of pneumococcal MEF isolations from children with recurrent AOM and AOM treatment failure [32, 35]. Presumably, in our study, this situation was directly related to antipneumococcal vaccination pursued only in 21% of tested children. There was a significantly lower frequency of pneumococcal colonization (0%) in immunized children both at the AOM (p = 0.015) and during post-treatment visits (p = 0.047). However, in one immunized patient S. pneumoniae isolate with serotype 6B was detected in MEF sample. A number of studies have examined the impact of antipneumococcal immunization with the use of conjugate vaccines on the burden of AOM. The reduction of visits for AOM, antibiotic prescription and tympanosomy tube placements were noted in these studies [13, 36]. However, in the Belgian study of children with the history of recurrent AOM immunized with PCV7 followed by a 23-valent polysaccharide booster, the authors reported no reduction in the number of AOM episodes and no changes in nasopharyngeal pneumococcal carriage during the 26-month follow-up period suggesting that the vaccine may not be useful when recurrent AOM is established [37].

After eight years since PCV7 introduction in United States it was reported that PCV7 serotypes virtually disappeared from MEF of vaccinated children with AOM [38]. High rate (73.7–80.1%) of PCVs serotype coverage of isolates colonizing upper respiratory tract in pre-school healthy children as well as in children with recurrent upper respiratory tract infections from south-east Poland was observed [20, 39]. According to Skoczynska et al. study from Poland [40], in children aged less than 5 years, the PCV10, and PCV13 covered 54.8%, and 68.8% of all IPD cases, and 76.3%, and 86.3% of cases involving children under 5 years of age. Our data showed that the S. pneumoniae serotype coverage by the currently available PCVs among isolates colonizing nasopharynx as well as otopathogens from MEF cultures in children with AOM from Poland is still very high (84–92%) and exceeded these reported in other European countries [35, 41]. A routine antipneumococcal vaccination has only been implemented in Poland in 2017 (https://gis.gov.pl/en/homepage/). Two years before, the PCVs had been recommended for children under 5 years old however with reimbursement only for sselected risk groups. National Institute of Public Health of National Institute of Hygiene annual data revealed slightly increasing percentage (from 1.5 to 4%) of vaccinated children aged 0–14 years in 2007–2014 period (http://wwwold.pzh.gov.pl/oldpage/epimeld/index_a.html).

In the present study, of the 10 clonal complexes recovered, all have previously been described in other countries, including 3 of the 43 worldwide spread resistant pneumococcal strains currently accepted by PMEN, Spain 9 V-3 (ST156), England 14–9 (ST9) and Spain 23F-1 (ST81). Spain23F-1 (ST81) and Spain9V-3 (ST156) have been present in Poland since the second half of the 1990s [2, 42]. A significant decrease in Spain23F-1 prevalence in some regions [43, 44] has been observed since the introduction of a seven-valent pneumococcal conjugate vaccine (PCV7) in 2001. Also in Poland the decrease of the Spain23F-1 clone occurrence from 19.1 to 6.8% among invasive and non-invasive PNSSP isolates was noticed [2, 42]. In our study, this clone constituted 10% of PNSSP isolates. Spain23F-1 is considered as a clone with low deposition of causing invasive diseases, and its adaptation to persistent colonization of the human nasopharynx may facilitate its intercontinental distribution [45]. The spreading of two related clones Spain9V-3 (ST156) and ST143, and their representatives seems to be responsible for the increase in resistance observed in 2002 in Poland [42]. Between 2002 and 2005, the Spain9V-3 complex spread in Poland from 22 to 47.5% of PNSSP isolates [2, 42]. In our study, CC1 (ST156/ST143/ST10340) isolates represented 30% of PNSSP isolates. Penicillin nonsusceptible variants of England14–9 (ST9) for the first time have been reported in Poland in 2004–2005 years [2] and in our study this clone with variants represented 20% of tested pneumococcal strains. Some of the previously detected clones [19] were found again, including ST87, ST135 and ST320 strains.

The high persistent prevalence of antibiotic-resistant S. pneumoniae strains in children with AOM after the completion of the therapy seems to confirm that the unsuccessful bacterial eradication may be regarded as a risk factor of infection recurrence. Presence of PCV serotypes as well as PMEN resistant clones in tested children are confirmation of their spreading caused by minimal uptake of PCVs in Polish population.