Nasopharyngeal Colonization and Antimicrobial Susceptibility of Bacterial Isolates in Children and Young Adults with Acute, Protracted, and Chronic Cough: A Cross-Sectional Bulgarian Study

Abstract

Since the nasopharynx serves as an ecological niche for Streptococcus pneumoniae, Corynebacterium spp., Haemophilus influenzae, Moraxella catarrhalis, etc., colonization is influenced by antimicrobial treatments, host immune responses, viral infections, and vaccines, often leading to local and systemic infections. We aimed to investigate the patterns of nasopharyngeal colonization and antimicrobial susceptibility of bacterial isolates in Bulgarian individuals under 20 years of age presenting with acute, protracted, and chronic cough. We analyzed 1383 samples using conventional culture methods, MALDI-TOF MS, antimicrobial susceptibility testing, and genetic analyses for Bordetella pertussis and Mycoplasma spp. Among 896 isolates, H. influenzae was the most prevalent (26.23%), followed by M. catarrhalis (23.55%), S. pneumoniae (22.54%), and S. pyogenes causing 7.59% of infections. In children (0–10 years), M. catarrhalis (198 isolates) and H. influenzae (142 isolates) were the most common pathogens, followed by S. pneumoniae (73 isolates), while in those aged 10–20 years, S. pneumoniae was the most common isolate (129), followed by H. influenzae (93) and M. pneumoniae (21). Colonization in children and young adults serves as a reservoir for pathogen transmission to adults, highlighting its significant public health implications. Monitoring bacterial colonization and resistance patterns remains essential to inform targeted prevention and treatment strategies.

1. Introduction

Respiratory tract infections, one of the most commonly reported infectious and communicable diseases, are the leading cause of childhood morbidity and mortality [1]. The nasopharynx, the upper part of the pharynx located behind the nasal cavity and above the soft palate, serves as an ecological niche for Gram-positive bacteria such as Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae, S. pyogenes, S. viridans group (e.g., S. mitis, S. sanguinis), Corynebacterium spp., and Actinomyces spp., as well as Gram-negative bacteria such as Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Neisseria meningitidis, Moraxella catarrhalis, Bordetella pertussis, Kingella kingae, Acinetobacter spp., Prevotella spp., Veillonella spp., and Capnocytophaga spp. [2,3]. However, particularly in children, these asymptomatic colonizers deals with selective pressure such as antimicrobial treatments, the host immune response, viral infections, and vaccines, and their carriage leads to local and systematic infections like epiglottitis, otitis media, bacteremia, sinusitis, pharyngitis, meningitis, tonsillitis, pneumonia, and pertussis [4]. Viral pathogens, including influenza A and B, respiratory syncytial virus (RSV), adenovirus, parainfluenza virus, and human bocavirus, account for 30–40% of respiratory tract infections [5]. SARS-CoV-2 accounted for 98.7% of respiratory virus cases in a study of over two million patients [6].

Children, especially those with chronic coughs lasting over eight weeks, play a significant role in the community’s spread of respiratory infections [7]. Nasopharyngeal colonizers, including S. pneumoniae, H. influenzae, and M. catarrhalis, are vital in shaping antimicrobial resistance (AMR) dynamics and infectious disease epidemiology [8]. These microorganisms are notable for their increasing resistance to commonly utilized antibiotics, facilitated by acquiring and disseminating antibiotic resistance genes (ARGs) [9]. The persistent emergence of multidrug-resistant (MDR) bacteria strains and the emergence of novel viral variants present significant challenges in treating chronic respiratory diseases and associated infections, with AMR predicted to result in 10 million annual deaths globally by 2050 [10]. In 2019, Bulgaria reported a significant burden of AMR, with approximately 1600 deaths directly attributable to AMR and an additional 6400 deaths associated with AMR, with E. coli and K. pneumoniae being the leading pathogens, commonly responsible for bloodstream infections, peritoneal and intra-abdominal infections, urinary tract infections, and pyelonephritis. The indirect deaths are often linked to secondary bacterial infections complicating viral illnesses, such as influenza and COVID-19, surgical site infections, and infections in immunocompromised individuals. All these data underscore the need to implement a national AMR action plan [11].

According to the World Health Organization (WHO), acute respiratory infections (ARIs) account for 4 million deaths annually, with a significant proportion of infants, children, and older adults [12]. In 2019, nearly 600 million cases of pneumonia and other lower respiratory tract infections (LRTIs) globally, as reported by the Global Burden of Disease Collaboration, resulted in 2.5 million casualties, especially among children under five from developing countries [13]. In 2014, pneumonia accounted for 80% of infectious disease-related deaths in the United States [14]. Since mid-2023, there has been a significant rise in pertussis cases across the EU/EEA, with 32,037 confirmed cases reported in the first quarter of 2024 [15]. Between January and June 2024, the UK Health Security Agency (UKHSA) recorded 10,493 confirmed pertussis cases, resulting in nine deaths [16].

This study, conducted from 3 January 2023 to 3 January 2024, aimed to investigate the patterns of nasopharyngeal colonization and antimicrobial susceptibility of bacterial isolates in Bulgarian individuals under 20 years of age presenting with acute, protracted, and chronic cough to understand the microbial profiles better and guide appropriate antibiotic therapy.

2. Materials and Methods

2.1. Subjects

From 3 January 2023 to 3 January 2024, a total of 1383 samples were submitted to the laboratory, including nasal and throat swabs, sputum, and sera from individuals under 20 years of age, presenting with acute, protracted, and chronic cough, distributed as follows: 48% female and 52% male. Among them, 872 tested positive. We tested nasal swabs (730), throat swabs (517), sputum samples (40), and sera for atypical bacterial pathogens (96). We selected individuals under 20 years of age to encompass the full range of pediatric and adolescent populations plus young adults presenting with prolonged cough and indicated for testing that aligned with the study objectives and design.

2.2. Methods

2.2.1. Identification of Isolates

Bacterial isolates were obtained from nasopharyngeal swabs collected using sterile techniques. The samples were inoculated onto appropriate selective and non-selective culture media, such as blood agar, chocolate agar, and MacConkey agar, and incubated under specific conditions (e.g., temperature and atmospheric requirements) to support the growth of target microorganisms. Colony morphology, hemolytic properties, and other preliminary observations were used to select colonies for further identification. Identification was then conducted using an automated microbial identification system based on innovative Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) Autof MS 1000 (Autobio Diagnostics, Zhengzhou, China).

2.2.2. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility of bacterial isolates was assessed using the disc diffusion method (DDM) following the Bauer–Kirby technique. Testing and interpretation were performed in accordance with the guidelines provided by The European Committee on Antimicrobial Susceptibility Testing (EUCAST) for the relevant year (www.eucast.org, accessed on 30 November 2024). Antibiotics tested included amoxicillin-clavulanic acid (20/10 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), azithromycin (15 µg), and others relevant to the pathogens identified. The categorization of susceptibility (S), resistance (R), and intermediate resistance (I) for the tested antibiotics was determined following the interpretative criteria established by the EUCAST guidelines. Antimicrobial susceptibility of bacterial isolates was assessed using the disc diffusion method (DDM) following the Bauer–Kirby technique. All disks were obtained from Oxoid Ltd., Basingstoke, Hampshire, UK, and potency of the disks were standardized using the reference strain E. coli ATCC 25922. Zone diameters measured via the disc diffusion method were compared with EUCAST breakpoints for each antibiotic and bacterial species. Isolates with zone diameters above the specified threshold were categorized as susceptible (S), while those below the resistance threshold were considered resistant. Intermediate isolates were defined as those with zone diameters falling between these thresholds, indicating reduced susceptibility that may still be treatable with adjusted dosing or at specific infection sites. We selected these antibiotics on the basis of their clinical relevance, local prescribing practices, and availability within Bulgaria, aiming to assess resistance profiles against commonly used therapies. If further details are required, we are happy to elaborate.

2.2.3. Gradient Diffusion Testing

Gradient diffusion tests were performed using the Epsilometer test (MIC Test Strip, Liofilchem, Roseto degli Abruzzi, Italy). The tests were conducted and interpreted according to the manufacturer’s recommendations and the latest EUCAST guidelines. The Minimum Inhibitory Concentration (MIC) was determined as the value where the elliptical growth edge intersected the strip. A single operator measured all MIC values. No duplicate isolates were used.

2.2.4. Genetic Analyses for Pertussis and Mycoplasma

Genetic analyses were performed to detect B. pertussis and M. pneumoniae in nasopharyngeal aspirates from individuals presenting with protracted cough or atypical pneumonia. B. pertussis DNA was identified using a BactoReal^® Kit Bordetella Multiplex (ingenetix GmbH; Haidingergasse 1, 1030 Vienna, Austria), a non-automated IVD test for the qualitative detection of DNA from B. pertussis, B. parapertussis, B. holmesii, and specific strains of B. bronchiseptica. The test targets the insertion sequence IS481 for B. pertussis, B. holmesii, and B. bronchiseptica, as well as IS1001 for B. parapertussis, B. bronchiseptica, and B. holmesii. A total of 110 samples (42 females and 68 males) were tested for B. pertussis, selected on the basis of the presentation of protracted cough. For M. pneumoniae, 237 samples from patients with atypical pneumonia were analyzed using VIASURE Real-Time PCR Detection Kits, targeting the CARDS gene for M. pneumoniae, the argR gene for Chlamydia pneumoniae, and the mip gene for Legionella pneumophila. These methodologies provided robust, high-sensitivity detection to evaluate the prevalence of these pathogens in the study population.

2.3. Ethical Considerations

The patients’ samples were submitted to the microbiology laboratory as part of their routine diagnostic testing. Informed consent was obtained from all individuals upon their agreement to provide samples for analysis before their ambulatory or hospital admission. According to our institutional guidelines, the study utilized anonymized data obtained from routine clinical diagnostics, which does not require formal Institutional Review Board (IRB) approval. However, this waiver was granted, following local ethical standards.

3. Results

We summarize the distribution of bacterial isolates collected from nasal, throat swabs, and sputum samples in individuals under 20 years of age, stratified by age groups (0–10 years and 10–20 years) in

Table 1. Etiological structure of bacterial isolates (overall data) and in children aged 0–10 years and aged 10–20 years.

Overall Data
Bacterial Species	Number of Isolates	%
Haemophilus influenzae	235	26.23
Moraxella catarrhalis	211	23.55
Streptococcus pneumoniae	202	22.54
Staphylococcus aureus	53	5.92
Streptococcus pyogenes	68	7.59
Haemophilus parainfluenzae	46	5.13
Mycoplasma pneumoniae (serology)	24	2.68
Corynebacterium spp.	19	2.12
Other	38	4.24
Total	896	100
Individuals aged 0–10 years
Bacterial species	Number of isolates
Haemophilus influenzae	142
Moraxella catarrhalis	198
Streptococcus pneumoniae	73
Staphylococcus aureus	38
Streptococcus pyogenes	45
Haemophilus parainfluenzae	28
Mycoplasma pneumoniae	3
Corynebacterium spp.	16
Other	16
Individuals aged 10–20 years
Bacterial species	Number of isolates
Haemophilus influenzae	93
Moraxella catarrhalis	13
Streptococcus pneumoniae	129
Staphylococcus aureus	15
Streptococcus pyogenes	23
Haemophilus parainfluenzae	18
Mycoplasma pneumoniae	21
Corynebacterium spp.	3
Other	22

. The table highlights the prevalence of bacterial pathogens in respiratory infections, especially in individuals with protracted, dry cough.

Among the 896 isolates identified, H. influenzae was the most prevalent pathogen, accounting for 26.23% of the total, followed closely by M. catarrhalis (23.55%) and S. pneumoniae (22.54%). S. pyogenes was responsible for 7.59% of infections, while Staphylococcus aureus accounted for 5.92%. H. parainfluenzae was less frequent, representing 5.13%.

In the 0–10 year age group, M. catarrhalis (198 isolates) and H. influenzae (142 isolates) were the most common pathogens, followed by S. pneumoniae (73 isolates). S. pyogenes and S. aureus were also identified at lower frequencies (45 and 38 isolates, respectively). In contrast, in the 10–20 year group, S. pneumoniae was the most common isolate (129), followed by H. influenzae (93) and M. pneumoniae (21). Interestingly, M. catarrhalis was less prevalent in this age group, with only 13 isolates. Other less common pathogens, such as S. pyogenes, H. parainfluenzae, and Corynebacterium spp., were also noted.

Our study assessed the antimicrobial susceptibility, resistance, and intermediate susceptibility percentages (S%, R%, and I%, respectively) of S. pneumoniae, M. catharralis, and H. influenzae in 1383 samples (nasal and throat swabs, and sputum) from individuals under 20 years of age, presenting with acute, protracted, and chronic cough. From the tested samples for pertussis, 51 were positive cases (23 females, 28 males), and for atypical pneumonia samples, there was a total of 237 (127 under 20 years old: 59 females, 68 males), of which 58 were positive cases (27 females, 31 males).

In

Table 2. Antimicrobial susceptibility of Streptococcus pneumoniae isolates (n = 202) to various antibiotics.

Streptococcus pneumoniae (n = 202)	S, %	R, %	I, %	MIC Range, µg/mL
Amoxicillin/clavulanic acid	83.53	4.12	12.35	0.016–8
Azithromycin	37.81	62.19	0.00	0.02−256
Cefpodoxime	99.38	0.62	0.00	0.016–0.5
Ceftriaxone	100.00	0.00	0.00	0.016–0.2
Cefuroxime	91.07	8.93	0.00	0.016–4
Clarithromycin	37.81	62.19	0.00
Clindamycin	56.21	43.79	0.00
Levofloxacin	89.22	4.19	6.59	<0.001–4
Moxifloxacin	100.00	0.00	0.00
Rifampicin	85.80	14.20	0.00

, we present 202 S. pneumoniae isolates, along with their corresponding MIC ranges (µg/mL). The data highlight the effectiveness of various antibiotics, including beta-lactams, macrolides, and fluoroquinolones, in treating S. pneumoniae infections, providing insight into current resistance patterns and therapeutic options.

We found high susceptibility (83.53%), with 4.12% isolates resistant to amoxicillin/clavulanic acid; full susceptibility (100%), indicating the continued efficacy of ceftriaxone and moxifloxacin; and high susceptibility for cefuroxime (91.07%), though resistance was noted in 8.93% of isolates. Both clarithromycin and clindamycin showed high resistance rates (62.19% and 43.79%, respectively), indicating limited efficacy against S. pneumoniae in this dataset. Clindamycin demonstrated a moderate susceptibility rate (56.21%), with 43.79% of isolates being resistant. Both levofloxacin and moxifloxacin maintained high susceptibility rates (89.22% and 100%, respectively) with minimal resistance. Rifampicin showed 85.80% susceptibility, though a notable resistance rate of 14.20% was observed.

In our study, 211 isolates of M. catarrhalis were tested for antimicrobial susceptibility. All isolates were 100% susceptible to amoxicillin/clavulanic acid and cefpodoxime, highlighting their continued effectiveness. Ampicillin resistance was universal, with 100% of isolates being resistant, consistent with the high prevalence of β-lactamase production, as confirmed by a positive cefinase test in all isolates (

Table 3. The proportion of bacterial isolates of M. catarrhalis susceptible to an antibiotic (susceptible percentage, S%) and the proportion of bacterial isolates resistant to an antibiotic (resistant percentage, R%).

Moraxella catarrhalis (n = 211)	S, %	R, %
Amoxicillin/clavulanic acid	100.00
Ampicillin	0.00	100.00
Azithromycin	75.33	24.67
Cefpodoxime	100.00
Cefuroxime	95.40	1.97
Clarithromycin	75.50	24.50
Clindamycin	58.82
Levofloxacin	99.33	0.67
Rifampicin	93.38	5.96
Trimethoprim/sulfamethoxazole	77.56	21.79
Producer of β-lactamase (positive cefinase test)	100%

).

Azithromycin and clarithromycin demonstrated moderate susceptibility rates (75.33% and 75.50%, respectively), with around one-quarter of the isolates being resistant to clarithromycin. Cefuroxime maintained high efficacy, with 95.40% susceptibility and only 1.97% resistance. Levofloxacin showed near-complete efficacy (99.33% susceptibility), while rifampicin and trimethoprim/sulfamethoxazole also demonstrated high activity, with susceptibility rates of 93.38% and 77.56%, respectively. Clindamycin exhibited a lower susceptibility rate of 58.82%, indicating reduced efficacy. Overall, the results underscore the effectiveness of β-lactam/β-lactamase inhibitor combinations and fluoroquinolones in treating M. catarrhalis infections while highlighting significant resistance to ampicillin and moderate resistance to macrolides and trimethoprim/sulfamethoxazole (Table 3).

Table 4. Antimicrobial susceptibility profile of Haemophilus influenzae (235 isolates).

Haemophilus influenzae (n = 235)	S, %	R, %	I, %	MIC Range, µg/mL
Moxicillin/clavulanic acid	68.44	9.02	22.54
Ampicillin	40.41	43.01	16.58	0.02–4
Ampicillin/sulbactam	69.58	9.17	21.25
Azithromycin	7.43	92.57	0.00
Cefixime	84.21	4.82	10.96	0.12–2
Cefpodoxime	97.86	1.28	0.85	0.05–0.5
Ceftriaxone	98.41	0.00	1.59	0.05–0.5
Cefuroxime	52.48	27.27	20.25	0.5–2
Ciprofloxacin	97.38	2.18	0.44	0.05–0.5
Clarithromycin	2.34	97.66	0.00
Levofloxacin	97.13	2.87	0.00	0.03–1
Moxifloxacin	98.33	0.00	1.67	0.03–0.5
Rifampicin	67.63	31.95	0.41	0.25–1.25
Trimethoprim/sulfamethoxazole	41.32	58.68	0.00
Producer of β-lactamase (positive cefinase test)	28%

presents the antimicrobial susceptibility, resistance, and intermediate susceptibility rates for 235 H. influenzae isolates and the corresponding MIC ranges (µg/mL). The results highlight the variable efficacy of commonly used antibiotics against H. influenzae and provide insight into current resistance patterns.

Beta-lactam antibiotics showed varied effectiveness, with 68.44% of isolates susceptible to amoxicillin/clavulanic acid and 69.58% to ampicillin/sulbactam. Notably, resistance to ampicillin was significant at 43.01%, consistent with the high percentage (28%) of beta-lactamase-producing isolates, as indicated by a positive cefinase test. Ceftriaxone and cefpodoxime demonstrated excellent efficacy, with susceptibility rates of 98.41% and 97.86%, respectively. Cefixime also showed high susceptibility at 84.21%, with minimal resistance (4.82%).

Among the fluoroquinolones, ciprofloxacin and levofloxacin retained strong activity, with susceptibility rates exceeding 97%, and moxifloxacin showed even higher susceptibility at 98.33%. Resistance to macrolides was striking, with only 7.43% of isolates being susceptible to azithromycin and 2.34% susceptible to clarithromycin, indicating widespread resistance. For rifampicin, susceptibility was moderate (67.63%), but 31.95% of isolates were resistant. Trimethoprim/sulfamethoxazole demonstrated limited efficacy, with 41.32% susceptibility and 58.68% resistance.

4. Discussion

In our study, we aimed to investigate the patterns of nasopharyngeal colonization and antimicrobial susceptibility of bacterial isolates of S. pneumoniae, M. catarrhalis, and H. influenzae in Bulgarian individuals under 20 years of age presenting with acute, protracted, and chronic cough. We were interested in assessing the prevalence of these bacteria in nasopharynx colonization since they play a critical role in acute respiratory tract infections, which is multifaceted, as they can act as primary pathogens or as opportunistic agents exacerbating viral infections. Epidemiological studies have shown that viral-bacterial co-infections significantly increase the severity and duration of respiratory infections, particularly in children [17,18].

In line with this, nasopharyngeal colonization by respiratory bacteria, such as S. pneumoniae and H. influenzae, plays a critical role in respiratory infections’ pathogenesis, often acting as a reservoir for invasive infections and influencing the course of illness. Antibiotic use remains a vital factor in managing these infections. However, the official data highlight regional variations in prescription practices across Europe and Bulgaria [19].

A concerning trend has emerged from pre- and post-pandemic analyses, which reveal shifts in the antimicrobial susceptibility patterns among common respiratory pathogens, emphasizing the importance of continuous surveillance to inform treatment guidelines and address antibiotic resistance [20]. Between 2019 and 2023, Bulgaria experienced a 27% increase in antibiotic consumption, sharply contrasting with the recommended 18% decrease by health organizations. This trend exacerbates the public health burden due to the growing risk of antibiotic resistance and highlights the urgent need for more effective measures to regulate antibiotic use [19,21].

Our results indicate a clear age-related distribution of bacterial pathogens in respiratory infections among individuals under 20 years of age. H. influenzae was the most prevalent bacterium overall, accounting for 26.23% of the total isolates. This is consistent with the findings in previous studies, highlighting the significant role of H. influenzae in respiratory infections, particularly in children [22]. In fact, H. influenzae was also the leading pathogen in the 0–10 year age group, where it accounted for 26.23% of the total isolates and was present in 142 of the 235 isolates in this category (Table 1). The high prevalence of H. influenzae in young children could be attributed to their underdeveloped immune systems and frequent exposure to this pathogen in environments such as daycare centers and schools [23]. Overall, these results highlight that while beta-lactam antibiotics and fluoroquinolones remain effective options, macrolides like azithromycin and clarithromycin show significant resistance, which may necessitate careful consideration in empirical treatment decisions.

M. catarrhalis, the second most common pathogen, was detected in 23.55% of the total isolates (Table 1), and it also showed high prevalence in both age groups, particularly in the 0–10 year group (198 isolates). This bacterium is a known cause of upper and lower respiratory tract infections in children, and its frequent appearance in nasal and throat samples aligns with previous literature. Its role as a colonizer in the nasopharyngeal region in children might explain its high isolation rates [24,25].

S. pneumoniae was another significant pathogen, identified in 22.54% of the total isolates (Table 1). However, a notable difference was observed in its prevalence across age groups. While S. pneumoniae was the most common pathogen in the 10–20-year-old group, with 129 isolates, it was less prevalent in the younger age group (73 isolates, Table 1). This shift in prevalence could reflect the protective effects of childhood immunization programs, as introducing pneumococcal vaccines has led to a decline in S. pneumoniae infections among younger children. However, as these children grow older and may be exposed to different strains or reduced vaccine coverage, the incidence of S. pneumoniae infections increases [26].

Interestingly, S. aureus and S. pyogenes showed lower prevalence compared with the top three pathogens, accounting for 5.92% and 7.59% of total isolates, respectively (Table 1). These pathogens were more common in the 0–10 age group, where S. pyogenes contributed to 45 isolates (Table 1). This finding suggests that while S. pyogenes and S. aureus may not be as dominant overall, they still play a role in respiratory infections in children, particularly in cases involving tonsillitis or pharyngitis [27,28].

The presence of H. parainfluenzae (5.13% of total isolates), M. pneumoniae (2.68% of total isolates), and Corynebacterium spp. (2.12% of total isolates) further supports the diversity of pathogens involved in respiratory infections. The lower isolation rates of these organisms may indicate that they are less common or less virulent than the more prominent bacteria like H. influenzae or S. pneumoniae. However, they remain important in specific patient populations, particularly in cases with atypical or resistant infections [29]. Overall, the results reveal substantial variability in antibiotic effectiveness, underscoring the need for careful antibiotic selection in treating H. influenzae infections, particularly in light of high macrolide resistance and beta-lactamase production.

In the 0–10 year group, the prevalence of M. pneumoniae was very low (3 isolates), but it was notably higher in the 10–20 year group (21 isolates), highlighting a shift towards atypical bacterial infections as children age. This is in line with the understanding that M. pneumoniae infections are more common in school-aged children and adolescents, as they are transmitted more easily in group settings. These results underscore the age-dependent variability in the prevalence of bacterial pathogens, with M. catarrhalis and H. influenzae dominating in younger children [30]. In comparison, Streptococcus pneumoniae was more common in older children and adolescents.

Lastly, it is worth noting that the category “other” includes pathogens that are less frequently encountered, contributing to 4.24% of the total isolates (Table 1). This category may encompass a range of uncommon or emerging pathogens that were not detected at the same frequency as the primary pathogens but may still play a role in certain clinical settings. These data emphasize the need for age-specific strategies in diagnosing and managing respiratory infections. The higher prevalence of H. influenzae and M. catarrhalis in younger children and the increasing frequency of S. pneumoniae in older children and adolescents calls for targeted antimicrobial therapy and vaccination programs [31]. Additionally, the presence of atypical pathogens like M. pneumoniae in the older age group underlines the importance of considering a broad differential diagnosis, especially in adolescents presenting with respiratory symptoms [32].

The clinical significance of MICs and MDR bacteria cannot be overstated, as they are critical indicators of the effectiveness of antibiotics and the severity of AMR. While this study did not include MIC determination or MDR-specific analyses, the existing literature underscores the alarming rise of MDR pathogens such as S. pneumoniae, H. influenzae, and M. catarrhalis, which pose significant challenges to treatment options. MIC data provide quantitative thresholds for guiding appropriate antimicrobial therapy, offering insights into the degree of resistance and potential treatment efficacy. The prevalence of MDR bacteria globally highlights the urgent need for continuous surveillance and robust diagnostic tools to guide antibiotic stewardship. Future research should aim to incorporate MIC-based analyses alongside traditional susceptibility testing to enable more detailed characterization of resistance patterns and inform clinical decision-making. Additionally, a deeper understanding of MDR mechanisms in nasopharyngeal isolates could enhance strategies to mitigate their transmission and impact on public health.

However, colonization in children is a significant reservoir for transmitting pathogens to adults, emphasizing its critical role in public health. Monitoring these trends is essential, as they provide valuable insights into bacterial spread dynamics and community resistance patterns. Understanding the role of pediatric colonization as a reservoir can inform prevention strategies and guide interventions to mitigate the risk of severe infections in more vulnerable populations. In line with this, recent advancements in antibiotic discovery have focused on addressing the growing challenge of AMR, which poses a significant threat to global public health [33]. Innovative approaches include the exploration of new antibiotic classes targeting bacterial metallophores, small molecules that are essential for bacterial metal acquisition and survival [34,35].

These efforts represent a promising strategy for developing targeted therapies with reduced off-target effects and slower resistance development. Additionally, the utilization of metals and metal-based compounds has emerged as a novel avenue to combat AMR, leveraging their unique properties to inhibit bacterial growth or enhance the efficacy of existing antibiotics. These strategies highlight a shift toward more specialized and mechanistic approaches in antibiotic discovery, aiming to outpace the rapid evolution of resistance in pathogens. Incorporating these advancements into clinical practice could significantly strengthen the arsenal against multidrug-resistant organisms, underscoring the critical need for continued research and investment in this area.

5. Conclusions

Our study demonstrated the diverse bacterial profiles associated with nasopharyngeal colonization in Bulgarian individuals under 20 years of age presenting with acute, protracted, and chronic cough. Among the 896 isolates identified, H. influenzae, M. catarrhalis, and S. pneumoniae emerged as the most prevalent pathogens, with distinct age-related variations in their distribution. The findings underscore the importance of understanding pediatric colonization as a critical reservoir for pathogen transmission, which plays a pivotal role in community health in Bulgaria.

Moreover, the observed antimicrobial susceptibility and resistance patterns provide crucial insights into the dynamics of bacterial spread and the selective pressures exerted by treatments and interventions. These data are essential for guiding appropriate antibiotic therapy and informing public health strategies to combat bacterial resistance and prevent severe infections in both children and adults. Continued monitoring of nasopharyngeal colonization trends and resistance patterns is vital to developing targeted interventions and safeguarding vulnerable populations.