Radiol Oncol 2025; 59(1): 147-152. doi: 10.2478/raon-2025-0018 147 research article Bronchial bacterial colonization and the susceptibility of isolated bacteria in patients with lung malignancy Sabrina Petrovic1, Bojana Beovic2,3, Viktorija Tomic3,4, Marko Bitenc1, Mateja Marc Malovrh3,4, Vladimir Dimitric4, Dane Luznik4, Martina Miklavcic1, Tamara Bozic1, Tina Gabrovec1, Aleksander Sadikov5, Ales Rozman3,4 1 Surgery Bitenc, Medical Centre Ljubljana (MCL), Ljubljana, Slovenia 2 Clinic for Infectious Diseases and Fever Conditions, University Medical Centre Ljubljana, Ljubljana, Slovenia 3 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 4 University Clinic of Pulmonary and Allergic Diseases Golnik, Golnik, Slovenia 4 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 5 Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2025; 59(1): 147-152. Received 15 April 2024 Accepted 19 January 2025 Correspondence to: Sabrina Petrovic, M.D., Kirurgija Bitenc d.o.o., Medical Centre Ljubljana (MCL), Vilharjev podhod 1, 1000 Ljubljana, Slovenia. E-mail: sabrina.petrovic@surgery-bitenc.com Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Postoperative pneumonia (POP) remains a leading cause of mortality following lung surgery. Recent studies have confirmed that the respiratory tract below the vocal cords is not sterile and often harbours potentially pathogenic microorganisms (PPMs), putting patients with lung malignancies at an increased risk for pulmonary infec- tions. Patients and methods. The study analysed 149 patients who underwent bronchoscopy for lung lesions suspected to be lung cancer. Protected specimen brush (PSB) samples were obtained during bronchoscopy prior to any specific treatment. Bacterial identification and antimicrobial susceptibility testing were conducted on the isolated strains. Results. Bacterial colonization was detected in 88.6% of patients, with 21.5% carrying PPMs. Notably, patients with type 2 diabetes exhibited a higher rate of PPM colonization compared to others. Antibiotic susceptibility testing showed no significant differences in efficacy between amoxicillin with clavulanic acid and first-generation cepha- losporin in both colonized patients and those with PPMs. Importantly, no multidrug-resistant bacteria were identified. Conclusions. Our findings indicate a slightly lower PPM colonization rate compared to previous studies, possibly due to the unique geographic characteristics of the study population. The absence of significant differences in bacterial susceptibility between the two tested antibiotics highlights the need for further research to refine perioperative infec- tion management strategies. Key words: bronchial bacterial colonization; potentially pathogenic microorganisms; antibiotic prophylaxis; lung cancer; bronchoscopy Introduction Postoperative pneumonia (POP) remains a sig- nificant contributor to postoperative mortality following lung surgery, with reported incidence rates ranging from 2% to 20%.1,2 Patients with lung malignancies are particularly susceptible to pul- monary infections due to factors such as immuno- suppression, impaired protective mechanisms, and localized inflammation caused by concurrent con- Radiol Oncol 2025; 59(1): 147-152. Petrovic S et al. Bacterial colonization in patients with lung malignancy148 ditions like bronchiectasis and chronic obstructive pulmonary disease (COPD).2 Recent studies have challenged the traditional belief that the respiratory tract below the vocal cords is sterile, highlighting the presence of mi- crobial colonization.3 However, limited research has focused on bronchial bacterial colonization (BBC) patterns in patients with lung malignan- cies. Existing studies report a wide range of BBC prevalence, from 10% to 83%, often involving po- tentially pathogenic microorganisms (PPMs) such as Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus.1,2,4,5 While the clinical significance of these microorganisms within the airways remains uncertain, their presence may influence the management and prognosis of lung cancer patients.3 Several risk factors, including age, gender, COPD, and smoking, have been asso- ciated with an increased likelihood of PPM colo- nization.1,2,4 Furthermore, studies have established a link between BBC and pneumonia in these pa- tients, though it remains unclear whether these bacteria contribute to postoperative infections af- ter lung surgery.1 Nevertheless, PPM colonization of the respiratory tract could elevate the risk of postoperative infections.2 The effectiveness of first-generation cephalo- sporins as perioperative antibiotic prophylaxis, as recommended by current guidelines, is under scrutiny due to the high incidence of postoperative pneumonia and the increasing prevalence of an- tibiotic-resistant bacteria among isolated strains.6-9 Addressing postoperative infections in patients with lung malignancies undergoing surgery is a critical clinical challenge, necessitating the identi- fication of effective prophylactic strategies. This study aims to prospectively evaluate the prevalence of PPM colonization in patients with lung malignancies, predominantly primary lung cancer, at the time of diagnosis before any specific treatment initiation. Additionally, it investigates antibiotic susceptibility among isolated bacteria to assess resistance rates and examines the potential association between PPM colonization and cancer stage. Patients and methods This prospective study was conducted from June 2021 to February 2023, focusing on patients pre- senting with lung lesions suspected to be primary lung cancer. During the initial outpatient evalu- ation, demographic and clinical data were col- lected, including age, gender, smoking history, and comorbidities. All patients were diagnosed following established guidelines for primary lung cancer diagnosis. TNM staging included chest, ab- dominal, and head CT scans, as well as PET-CT imaging. Flexible bronchoscopy was performed for all patients to obtain tumour tissue samples for histological diagnosis when possible. In addition, protected specimen brush (PSB) samples were col- lected during bronchoscopy prior to initiating any specific treatment. For cases where bronchoscopic tumour r access was not feasible, CT-guided needle biopsies were used to determine histological typ- ing. PSB samples were sent to the microbiology labo- ratory, where bacterial colonization was defined as the isolation of microorganisms at a threshold of ≥10³ CFU/mL. Antimicrobial susceptibility testing was performed on each bacterial isolate using the microbiology protocol tailored to the bacterial spe- cies. The study received approval from the National Medical Ethics Committee of the Republic of Slovenia (no. 0120-163/2021/3), and all participants provided written informed consent. TABLE 1. Baseline characteristics of patients Characteristics n % Patients 149 Male 90 60.4 Median age (years) 66 Smokers 50 33.6 Ex-smokers 71 47,7 Non-smokers 28 18,8 COPD 44 29.5 Diabetes type 2 13 8.7 Colonized patients 132 88.6 Colonized with PPMs 32 21.5 Multiple bacteria colonization 86 57.7 Adenocarcinoma 86 57.7 Squamous cell carcinoma 22 14.8 Small cell carcinoma, carcinoid or large cell carcinoma 11 7.4 Non-small cell carcinoma NOS* 17 11.4 Other, non-lung cancer malignancies (limfoma, methastases) 13 8,7 COPD = chronic obstructive pulmonary disease; NOS = not otherwise specified; PPMs = potentially pathogenic microorganisms Radiol Oncol 2025; 59(1): 147-152. Petrovic S et al. Bacterial colonization in patients with lung malignancy 149 Bronchoscopy Bronchoscopy was performed under moderate sedation, adhering to a strict no-suction policy prior to reaching the carina. Upon entering the trachea, topical lidocaine anaesthesia was admin- istered to the main and upper lobar bronchi. Sterile brushes (OLYMPUS disposable cytology brush BC-202D-210) were used to collect samples from the bronchi of the tumour-bearing lobe prior to di- agnostic sampling to detect bacterial colonization. Each sample was preserved in 1 mL of sterile saline solution and sent to the microbiology laboratory. Peripheral tumour sampling was conducted using various bronchoscopic techniques to determine tu- mour histological types. Microbiological analysis PSB samples were promptly processed in the mi- crobiology laboratory. Samples were vortexed, and slides were prepared before dilution and plating. Gram staining and microscopic examination as- sessed sample quality, bacterial morphology, and abundance. Samples were diluted to a final concen- tration of 10⁻³ and inoculated on various solid and liquid media, including blood agar, chocolate agar, Brucella blood agar, CHROMagar™ Orientation (CHROMagar, France), and thioglycollate broth. Plates were incubated aerobically and anaerobi- cally at 35°C and evaluated for growth at 24, 48, and 72 hours. Liquid medium subculturing onto the same solid media plates confirmed bacterial morphotypes and colony-forming units per milli- litre (CFU/mL). A threshold of ≥10³ CFU/mL was used to define positive culture results. Bacterial identification and antimicrobial sus- ceptibility testing were performed using the MALDI Biotyper® (Bruker Daltonics GmbH & Co, Germany) and the standardized EUCAST disc dif- fusion method. Bacteria were classified as PPMs (e.g., S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus, P. aeruginosa, Enterobacterales) or non-PPMs (e.g., Streptococcus viridans group, Neisseria spp., Corynebacterium spp., coagulase-negative staphylo- cocci).5 Statistical analyses Descriptive statistics were presented as median (range) for continuous variables and as frequen- cies and proportions for categorical variables. Comparisons of bacterial colonization rates with respect to tumour stage and comorbidities, as well TABLE 2. Number and percentage of recovered bacteria RECOVERED BACTERIA No. of patients with isolated species % of patients with isolated species Streptococcus mitis 53 35,6% Streptococcus salivarius 36 24,2% Streptococcus oralis 27 18,1% Streptococcus parasanguinis 23 15,4% Streptococcus vestibularis 18 12,1% Veillonella atypica 13 8,7% Haemophilus influenzae 12 8,1% Streptococcus pneumoniae 11 7,4% Neisseria subflava 9 6,0% Actinomyces odontolyticus 9 6,0% Staphylococcus aureus 8 5,4% Haemophilus parahaemolyticus 8 5,4% Streptococcus gordonii 8 5,4% Rothia mucilaginosa 7 4,7% Escherichia coli 6 4,0% Staphylococcus epidermidis 6 4,0% Staphylococcus hominis 4 2,7% Streptococcus anginosus 4 2,7% Veillonella parvula 3 2,0% Fusobacterium periodonticum 3 2,0% Moraxella catarrhalis 2 1,3% Pseudomonas aeruginosa 2 1,3% Haemophilus parainfluenzae 2 1,3% Corynebacterium simulans 2 1,3% Prevotella nigrescens 2 1,3% Streptococcus constellatus 2 1,3% Gemella haemolysans 2 1,3% Serratia marcescens 2 1,3% Prevotella melaninogenica 2 1,3% Granulicatella adiacens 2 1,3% Streptococcus agalactiae 1 0,7% Staphylococcus capitis 1 0,7% Streptococcus cristatus 1 0,7% Neisseria macacae 1 0,7% Neisseria cinerea 1 0,7% Neisseria flavescens 1 0,7% Veillonella dispar 1 0,7% Prevotella jejuni 1 0,7% Campylobacter concisus 1 0,7% Citrobacter koseri 1 0,7% Prevotella pallens 1 0,7% Enterobacter bugandensis 1 0,7% Acinetobacter lwoffii 1 0,7% Moraxella nonliquefaciens 1 0,7% Radiol Oncol 2025; 59(1): 147-152. Petrovic S et al. Bacterial colonization in patients with lung malignancy150 as antibiotic susceptibility, were assessed using Pearson’s chi-squared test or Fisher’s exact test, as appropriate. A p-value < 0.05 was considered sta- tistically significant. All p-values are two-tailed. Statistical analyses were conducted using IBM SPSS (version 21, Chicago, IL, USA). Results The study included 149 consecutive patients with lung malignancies, with a median age of 66 years (20–84). Baseline characteristics of the participants are summarized in Table 1. Most patients (71.8%) were diagnosed with non-small cell lung cancer, primarily adenocarcinoma (57%). Respiratory tract colonization with at least one bacterial strain was confirmed in 132 patients (88.6%), with 86 patients (57.7%) harbouring multi- ple bacterial strains. Colonization with potentially pathogenic microorganisms (PPMs) was identi- fied in 32 patients (21.5%). Antibiotic sensitivity testing for amoxicillin with clavulanic acid and first-generation cephalosporins was performed in 120 patients. Sensitivity testing for amoxicillin with clavulanic acid and first-generation cephalo- sporins was not conducted for 12 patients due to colonization with bacteria requiring specific an- tibiotic panels (Rothia mucilaginosa, Streptococcus constellatus, Actinomyces odontolyticus, Streptococcus cristatus, and Fusobacterium periodonticum), none of which were classified as PPMs. The most frequently isolated PPMs were Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli (Table 2), while the most common non-PPMs included Streptococcus mitis and Streptococcus salivarius. Notably, 57.7% of patients exhibited colonization by multiple bacterial strains. No statistically significant differences in PPM colonization rates were observed across different cancer stages (Table 3). Similarly, no significant association was found between COPD and colo- nization with potentially pathogenic bacteria (p = 0.39) (Table 4). However, type 2 diabetes emerged as an independent risk factor for colonization with potentially pathogenic bacteria (p = 0.04) (Table 5). Antibiotic susceptibility testing revealed no significant differences in efficacy between amoxi- cillin with clavulanic acid and first-generation cephalosporin in both colonized patients and those colonized specifically by PPMs (Tables 6 and 7). TABLE 3. Relationship between cancer stage and colonization with potentially pathogenic microorganisms (PPMs) STAGE (8th TNM classification) PPMs Total no yes I 50 11 61 82.0% 18.0% 100.0% II 25 7 32 78.1% 21.9% 100.0% III 16 6 22 72.7% 27.3% 100.0% IV 11 2 13 84.6% 15.4% 100.0% Total 102 26 128* 79.7% 20.3% 100.0% *for patients, who didn’t have primary lung cancer, cTNM was not defined TABLE 4. Relationship between colonization with potentially pathogenic microorganisms (PPMs) and chronic obstructive pulmonary disease (COPD) COPD PPMs Total no yes no 83 21 104 79.8% 20.2% 100.0% yes 32 12 44 72.7% 27.3% 100.0% Total 115 33 148* 77.7% 22.3% 100.0% *for 1 patient, there was no comorbidity data TABLE 5. Relationship between colonization with potentially pathogenic microorganisms (PPMs) and diabetes type 2 DIABETES TYPE 2 PPMs Total no yes no 108 27 135 80.0% 20.0% 100.0% yes 7 6 13 53.8% 46.2% 100.0% Total 115 33 148* 77.7% 22.3% 100.0% *for 1 patient, there was no comorbidity data Radiol Oncol 2025; 59(1): 147-152. Petrovic S et al. Bacterial colonization in patients with lung malignancy 151 Discussion In this study, we conducted a prospective inves- tigation of BBC in patients suspected of primary lung cancer before initiating any treatment. Our methodology introduced a key distinction from previous studies by using sterile brush specimens to collect samples from the bronchi of the tumour- containing lobe. Additionally, we evaluated the an- tibiotic susceptibility of isolated bacteria to antibi- otics commonly used for perioperative prophylaxis in thoracic surgery. Our findings revealed a lower prevalence of colonization by PPMs (21.5%) compared to previ- ous studies. Only two patients harboured bacte- ria resistant to both amoxicillin with clavulanic acid and first-generation cephalosporin. In one in- stance, bacteria were resistant to amoxicillin with clavulanic acid but susceptible to first-generation cephalosporin, while the reverse was observed in another case. Importantly, there were no signifi- cant differences in susceptibility between the two antibiotics, and no multidrug-resistant bacteria were identified. In a similar study, Laroumagne et al. examined bronchial colonization at the time of lung cancer diagnosis. They reported a higher prevalence of PPM colonization (50%), likely due to non-sterile sampling conditions. Their findings suggested an association between bronchial colonization and lower survival rates, potentially linked to infec- tious complications.4 Ioanas et al. reported a PPM colonization rate of 41%, again using non-sterile sampling techniques. Their study demonstrated no resistance to con- ventional antibiotics, consistent with our findings. They also reported a low incidence of postopera- tive pulmonary infections (12%) and no pneumo- nia cases, likely attributable to effective prophy- laxis with first-generation cephalosporin adminis- tered perioperatively and for 48 hours postopera- tively. Similar complication rates were observed in colonized and non-colonized patients, although their study was limited to 41 patients.5 Dancewicz et al. also reported similar BBC rates and found no evidence of multidrug-resistant mi- croorganisms, aligning with our results.2 Boldt et al., however, reported a PPM colonization rate of 48% in patients undergoing lung surgery. They found that a single dose of sulbactam plus ampi- cillin was significantly more effective than first- generation cephalosporin in preventing infec- tions, suggesting alternative regimens for prophy- laxis.10 Radu et al. conducted a retrospective analysis of 312 cases, highlighting the inefficacy of first- generation cephalosporin in 84% of cases, raising concerns about current prophylactic guidelines.8 Schlusser et al. suggested that targeted antibiotic prophylaxis against bronchial colonizing bacteria could reduce postoperative pneumonia incidence. They observed a significant reduction when an- tibiotics were tailored to the identified bacteria, though their study was not randomized and war- rants further validation.6,7 Lastly, D’Journo et al.’s meta-analysis established a statistical correlation between preoperative BBC and postoperative respiratory complications, em- phasizing the clinical importance of preoperative colonization screening.1 Conclusions This study provides valuable insights into bron- chial bacterial colonization in patients with lung malignancies, predominantly primary lung cancer. The prevalence of PPM colonization and the low resistance to tested antibiotics characterize a pa- tient population primarily from central and west- ern Slovenia, differing from studies conducted in other geographical regions. While PPM coloniza- TABLE 6. Susceptibility among all colonized patients Amoxicillin with clavulanic acid First generation cephalosporin Total R S S/R R 2 2 0 4 S 2 101 1 104 S/R 0 6 6 12 Total 4 109 7 120 R = resistant; S = susceptible TABLE 7. Susceptibility among patients colonized by potentially pathogenic microorganisms (PPMs) Amoxicillin with clavulanic acid First generation cephalosporin Total R S S/R R 1 1 0 2 S 1 20 0 21 S/R 0 5 3 8 Total 2 26 3 31 R = resistant; S = susceptible Radiol Oncol 2025; 59(1): 147-152. Petrovic S et al. Bacterial colonization in patients with lung malignancy152 tion was not associated with lung cancer stage or COPD, a significantly higher prevalence was ob- served in patients with type 2 diabetes. The absence of significant differences in an- tibiotic susceptibility between amoxicillin with clavulanic acid and first-generation cephalosporin highlights the need for further research. Given the substantial rates of colonization and postoperative pneumonia, we recommend routine microbiologi- cal sampling during bronchoscopy for all patients suspected of primary lung cancer. This approach could enable targeted perioperative antibiotic prophylaxis in patients undergoing thoracic sur- gery. Future prospective studies comparing tar- geted versus standard prophylaxis are essential to establish best practices. References 1. D’Journo XB, Rolain JM, Doddoli C, Raoult D, Thomas PA. Airways coloniza- tions in patients undergoing lung cancer surgery. Eur J Cardiothorac Surg 2011; 40: 309-21. doi: 10.1016/j.ejcts.2010.11.036 2. Dancewicz M, Szymankiewicz M, Bella M, Sviniarska J, Kowalewski J. [Bronchial bacterial colonization in patients with lung cancer]. [Polish]. Pneumol Alergol Pol 2009; 77: 242-7. PMID: 9591094 3. Prat C, Lacoma A. Bacteria in the respiratory tract – how to treat? Or do not treat? Int J Infect Dis 2016; 51: 113-22. doi: 10.1016/j.ijid.2016.09.005 4. 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