1 Volume 24 2025 e257686 Original Research Braz J Oral Sci. 2025;24:e257686http://dx.doi.org/10.20396/bjos.v24i00.8677686 1 Experimental Biology Post-Graduate Program (PGBIOEXP), Federal University of Rondônia (UNIR) and Oswaldo Cruz Foundation (Fiocruz), Porto Velho, Rondônia, Brazil. 2 Microbiology Laboratory, Oswaldo Cruz Foundation (Fiocruz), Porto Velho, Rondônia, Brazil. 3 Oswaldo Cruz Foundation (Fiocruz), Porto Velho, Rondônia, Brazil. 4 Tropical Medicine Research Center (CEPEM), Porto Velho, Rondônia, Brazil. Corresponding author: Najla Benevides Matos, Public Heath Reseacher, Microbiology Laboratory, Fundação Oswaldo Cruz Rondônia Rua da Beira, nº 7671, Lagoa, CEP: 76812-245, Porto Velho, Rondônia, Brazil. Phone number: +55 69 993729393. E-mail: najla.matos@fiocruz.br Editor: Dr. Altair A. Del Bel Cury Received: September 03, 2023 Accepted: November 12, 2024 Oral cavity colonization by Pseudomonas aeruginosa in patients admitted in the intensive care units (ICUs) Marcos Eduardo Passos da Silva1 , Luccas Manoel de Melo Suica2 , Renata Santos Rodrigues3 , Izabelly Vitória Gotara Ramos2 , Anjo Gabriel Carvalho1 , Nucia Cristiane da Silva Lima4 , Mayra Gyovana Leite Belém2 , Rosimar Pires Esquerdo3 , Najla Benevides Matos3* Patients admitted in the Intensive Care Units (ICUs) often experience various oral health issues during their hospitalization, which can lead to a shift in their oral flora from Gram-positive to pathogenic Gram-negative bacteria, including Pseudomonas aeruginosa. Aim: This study aimed to identify the colonization of P. aeruginosa in the oral cavity of ICU patients, as well as to assess its antimicrobial susceptibility profile, phenotypic resistance mechanisms, and biofilm-forming capacity. Methods: Oral cavity samples were collected from patients in ICUs across three public hospitals in Porto Velho, Rondônia, Brazil. Antimicrobial susceptibility testing was performed using the disk diffusion method, phenotypic research on resistance determinants was conducted using a disk method with and without the addition of enzymatic inhibitors, and biofilm formation was assessed by measuring absorbance in microplates. Results: A total of 114 oral cavity samples were obtained, with 28% showing colonization by P. aeruginosa. The antimicrobial susceptibility profile revealed that 93.7% of the isolates were susceptible to Polymyxin B. The highest rate of non-susceptibility was observed with levofloxacin (46.8%), followed by carbapenems: imipenem (43.7%) and meropenem (40.6%). Additionally, 37.5% of the isolates were classified as multidrug-resistant (MDR). In phenotypic research for carbapenemases, 35.7% of the isolates were positive for plasmid-mediated AmpC, and 14.3% were positive for Metallo-β-lactamases. Regarding biofilm production, 75% of the isolates were classified as moderate/strong biofilm producers. Conclusion: Therefore, oral cavity colonization by P. aeruginosa in ICU patients is evident, with notable multidrug resistance to antibiotics, highlighting the need for vigilant monitoring and effective oral hygiene practices for these patients. Keywords: Biofilms. Bacteria. Drug resistance. https://orcid.org/0000-0002-4973-0181 https://orcid.org/0009-0004-2135-1368 https://orcid.org/0000-0001-7954-864X https://orcid.org/0009-0001-2452-8569 https://orcid.org/0000-0002-1870-0465 https://orcid.org/0000-0001-8588-3188 https://orcid.org/0000-0002-1801-4115 https://orcid.org/0009-0007-3227-4662 https://orcid.org/0000-0002-7271-5764 2 Silva et al. Braz J Oral Sci. 2025;24:e257686 Introduction The human microbiome is a critical factor in maintaining the balance between health and disease, with the oral microbiota being the second most diverse micro- bial community after the intestinal microbiota. It serves as a primary entry point for various microorganisms1. According to the Human Oral Microbiome Data- base, there are over 774 oral bacterial species, with 58% of these already identified and named. In a healthy individual, a balanced and stable oral microbiota acts as a barrier that pre- vents the colonization of opportunistic pathogens and subsequent oral or systemic infections2. However, an imbalance between the microbiota and the host can lead to the onset of various pathologies, such as periodontal diseases, tonsillitis, systemic infections, cardiovascular diseases, and pneumonias3. Patients admitted to an Intensive Care Unit (ICU) often experience various oral health issues caused by factors such as malnutrition, endotracheal tubes, reduced fluid intake, and oral medication administration. These factors, combined with inadequate oral care, lead to oral mucosal dryness and inflammation, altered sali- vary flow, increased dental plaque formation, and biofilm accumulation with patho- genic bacteria4,5. Consequently, within the first 48 hours after ICU admission, the patient’s oral flora is likely to change, being predominantly replaced by pathogenic Gram-negative bacteria such as Pseudomonas aeruginosa, which is mainly associ- ated with Ventilator-Associated Pneumonia (VAP)6. P. aeruginosa is a Gram-negative, ubiquitous bacillus found in various environments such as water, soil, food, and hospital settings7. It is an opportunistic pathogen responsible for Healthcare-Associated Infections (HAIs) in ICUs, primarily affecting immunocompromised patients, those with pulmonary infections, and patients with cystic fibrosis, leading to high morbidity and mortality rates8,9. Ventilator-Associated Pneumonia (VAP) is a pulmonary infection that develops after 48 hours of patient hospitalization, accounting for more than 20% of hospital-acquired infections10. P. aeruginosa is a leading cause of these infections, with mortality rates exceeding 30%11. Furthermore, the widespread emergence of multidrug-resistant strains of this pathogen has become a global public health issue, impacting clinical outcomes and increasing morbidity and mortality rates within ICUs12. The World Health Organization13 (WHO, 2024) classifies carbapenem-resistant P. aeruginosa (CRPA) as a high group pathogen for which research and development of new antibiotics are urgently needed due to the extensive resistance of this bacte- rium to antimicrobials. Resistance to carbapenems has become a health emergency, as this class of antibiotics represents a high-potency, broad-spectrum option used as a last resort for infections caused by this species14. P. aeruginosa has various mechanisms of antibiotic resistance related to membrane permeability, such as efflux pumps and reduced production of porins, as well as the production of enzymes that hydrolyze antibiotics, including carbapenemases15. The 3 Silva et al. Braz J Oral Sci. 2025;24:e257686 emergence and spread of carbapenemases have become a serious issue, as they confer resistance not only to carbapenems but also to other β-lactam antibiotics and β-lactamase inhibitors16. A global cohort study conducted by Reyes et al.17 (2023) to analyze the global fre- quency and clinical impact of CRPA found that South and Central America had the highest rate of carbapenemase presence, with 69% of isolates. Additionally, data from the SENTRY program reveal that between 1997 and 2016, P. aeruginosa had a 44.6% isolation rate in hospitalized patients, with a high tendency towards resis- tance to carbapenems18. Bacterial biofilms are also associated with antibiotic resistance in P. aeruginosa. In addition to being an intrinsic biofilm producer, biofilm formation is consid- ered a virulence factor for the species, enhancing its ability to colonize and resist extreme environments and antibiotics19. Inadequate oral hygiene in ICU patients leads to increased dental plaque (biofilm), which becomes a reservoir for patho- genic microorganisms, increasing plaque mass and producing more biofilm. This, in turn, reinforces colonization and serves as an entry point for infections in the host4,6. Therefore, considering the prevalence, pathogenic role, and high morbidity and mor- tality rates caused by P. aeruginosa in ICU patients, this study aimed to investigate the colonization, antimicrobial susceptibility profile, biofilm formation, and phenotypic detection of carbapenemases in isolates of this pathogen derived from oral cavity samples of patients admitted to ICUs. Materials and Methods Sample Collection and Processing Samples were collected between December 2017 and February 2018, and between December 2018 and January 2019. Oral cavity secretions were obtained using swabs from patients hospitalized in three public hospitals (referred to as Hospital I, Hospital II, and Hospital III) in Porto Velho, Rondônia, Brazil. Following collection, the samples were transported to the Microbiology Laboratory at Fiocruz – Rondônia in thermally controlled containers for processing, isolation, identification, and charac- terization of microorganisms. This study was approved by the Ethics Committee of Tropical Medicine Research Center, Porto Velho, Rondônia, Brazil (Process n. 2.368.951). Bacteriology and Isolation of Isolates All samples were inoculated onto various culture media, including Blood Agar (BA; HiMedia®, India), MacConkey Agar (MC; FirstLab®, Brazil), Cetrimide Agar (CT; Biolog®), and Luria-Bertani Broth (LB; Kasvi®, Spain). The plates were then incubated in bacterial incubators at approximately 37°C for 18 to 24 hours. Colonies suggestive of P. aeruginosa were subjected to genomic DNA extraction using phenol-chloroform, followed by amplification of the 16S rRNA gene using conven- 4 Silva et al. Braz J Oral Sci. 2025;24:e257686 tional PCR as described by Arruda et al.20. The amplified products were purified using the QIAquick Gel Extraction Kit (QIAGEN®, Germany) according to the manufacturer’s protocol. The purified DNA was quantified using NanoDrop1000® (Thermo Scien- tific®, USA) and sequenced using Sanger methodology. Sequence analysis and consensus sequence generation were performed using BioEdit Sequence Alignment Editor (version 7.0), and species identification and confirmation were carried out using the Basic Local Alignment Search Tool (BLAST) database. Antimicrobial Susceptibility Testing Antimicrobial susceptibility testing was conducted using the disk diffusion method on Mueller-Hinton Agar (Kasvi®, Italy), following the Clinical Laboratory Stan- dards Institute (CLSI) 2022 guidelines. The following antibiotics were tested: Piperacillin-tazobactam (TTP – 100/10 µg), ceftazidime-avibactam (CZA – 30/20 µg), ceftolozane-tazobactam (C/T – 30/10 µg), ceftazidime (CAZ – 30 µg), cefepime (CPM – 30 µg), aztreonam (ATM – 30 µg), imipenem (IPM – 10 µg), meropenem (MER – 10 µg), gentamicin (GEN – 10 µg), tobramycin (TOB – 10 µg), amikacin (AMI – 30 µg), ciprofloxacin (CIP – 5 µg), and levofloxacin (LVX – 5 µg). E. coli ATCC® 25922 was used as a quality control. Multidrug resistance (MDR) was classified according to the criteria established by Magiorakos et al.21. For susceptibility testing to Polymyxin B, the microdilution method was used to determine the minimum inhibitory concentration (MIC) according to the POLICIM- BAC kit (Probac®, Brazil). Pseudomonas aeruginosa ATCC® 27853 was used as a quality control. Phenotypic Detection of Carbapenemases Phenotypic detection of carbapenemases was performed following the method- ology outlined in Technical Note No. 01/2013 from the Brazilian National Health Surveillance Agency (ANVISA). A combined disk test was conducted using ertap- enem (ETP – 10 µg), imipenem (IPM – 10 µg), and meropenem (MER – 10 µg) with and without the addition of enzyme inhibitors. For enzyme interpretation and classification: for Ambler class A, IPM and MER were impregnated with phenylbo- ronic acid (PBA); for class B, ethylenediaminetetraacetic acid (EDTA) was used; and for class C, PBA or cloxacillin (CLOXA) was used. Quality control for the tests included E. coli ATCC® 25922, Klebsiella pneumoniae CCBH 16302, Klebsiella pneumoniae CCBH 6556, and other strains from the CCBH collection at Fiocruz (http://ccbh.fiocruz.br/). Biofilm Formation Test The biofilm formation capacity of the isolates was evaluated in triplicate as described by Alvim et al.22 with modifications, using 96-well polystyrene microplates. P. aeruginosa isolates were grown in Luria-Bertani broth (LB; Kasvi®, Spain) in an orbital shaker at 120 rpm at approximately 37°C overnight for 18 to 24 hours. After this period, the culture was diluted 1:20 in sterile LB, and 200 µl of the suspen- http://ccbh.fiocruz.br/ 5 Silva et al. Braz J Oral Sci. 2025;24:e257686 sion was transferred in triplicate to the 96-well polystyrene microplates (Costar, USA), which were incubated at approximately 37°C for 24 hours. Subsequently, the plates were washed twice with 200 µl of distilled water to remove LB and non-ad- hered bacteria. Adhered bacteria were then stained for 5 minutes with 100 µl of crystal violet (0.1% w/v, Laborclin®, Brazil), excess dye was removed with distilled water, and elution was performed with 95% ethanol (Neon®, Brazil). Biomass was quantified by measuring absorbance with a spectrophotometer (Biotek Epoch®, USA) at a wavelength of 570 nm (optical density – OD). E. coli ATCC® 25922 was used as a negative control, and P. aeruginosa ATCC® 27853 was used as a positive control. Results were interpreted based on the average of triplicates for each isolate. The cut- off point was calculated by the mean OD of the negative control plus three times the standard deviation of the same. Isolates with OD values equal to or below the cutoff point were considered weak/non-biofilm producers, while those with OD values above the cutoff point were considered moderate/strong biofilm producers. Statistical Analysis Statistical analyses were performed using GraphPad Prism 5.0 software, employing non-parametric tests such as Fisher’s Exact Test and Odds Ratio. Statistical signifi- cance was defined as a p-value < 0.05. Results A total of 144 oral cavity samples were collected. Stratified by hospital, 42.1% (48/114) of the samples were from Hospital I, 32.4% (37/114) from Hospital III, and 25.5% (29/114) from Hospital II. Of these, 28% (32/114) of the patients were found to be colonized by Pseudomonas aeruginosa. The antibiotic susceptibility profile showed that 93.7% (30/32) of the oral cavity iso- lates were susceptible to polymyxin B, 84.3% (27/32) to piperacillin-tazobactam, 81.2% (26/32) to ceftazidime-avibactam and ceftolozane-tazobactam, and 78.1% (25/32) to ceftazidime and cefepime. Among the non-susceptible isolates, the highest rate observed was for levofloxacin at 46.8% (15/32), followed by carbapenems, with 43.7% (14/32) for imipenem and 40.6% (13/32) for meropenem. Additionally, two iso- lates exhibited resistance to polymyxin B. Figure 1 illustrates the susceptibility profile of the oral cavity isolates to all tested antibiotics. In terms of multidrug resistance, 37.5% (12/32) were classified as MDR. 6 Silva et al. Braz J Oral Sci. 2025;24:e257686 0 5 10 15 20 25 30 35 CZA N um be r o f i so la te s Antibiotics Susceptible Non-susceptible PBLVXCIPAMITOBGENMERIPMATMCPMCAZPPTC/T AMI: amikacin; ATM: Aztreonam; CAZ: Ceftazidime; CIP; Ciprofloxacin; CPM: Cefepime; CZA: Ceftolozane-Avibactam; C/T: Ceftazidime-Tazobactam; GEN: Gentamicin; IPM: Imipenem; LVX: Levofloxacin; MER: Meropenem; PB: Polymyxin B; PPT: Piperacillin-Tazobactam; TOB: Tobramycin. Figure 1. Antimicrobial susceptibility profile of Pseudomoas aeruginosa isolates from the oral cavity of patientes admitted in the ICUs. For the phenotypic detection of carbapenemases, only isolates that exhibited non-susceptibility to carbapenems (imipenem and meropenem) were tested. Thus, 14 oral cavity isolates were assessed, with 50% (7/14) being negative, 35.7% (5/14) positive for plasmid-mediated AmpC, and 14.3% (2/14) positive for metallo-β-lactamases (MBL). Table 1 presents the quantity of detected enzymes and the resistance classification of the isolates. Table 1. Phenotypic detection of carbapenemases and multidrug resistance classification in Pseudomonas aeruginosa isolates from the oral cavity of patients admitted in the ICUS. Oral cavity Resistance classification carbapenemases MDR Non-MDR total Plasmid-mediated AmpC 60% (3/5) 40% (2/5) 100% (5/5) MBL 100% (2/2) 0 100% (2/2) Negative 71,4% (5/7) 28,6% (2/7) 100% (7/7) Biofilm production was observed in 75% (24/32) of the oral cavity isolates as moderate/strong biofilm producers, while 25% (8/32) were weak/non-producers. Figure 2 displays the average absorbance obtained for each tested isolate. 7 Silva et al. Braz J Oral Sci. 2025;24:e257686 O D 57 0 nm 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Biofilm forming from oral cavity isolates Non-biofilm forming Biofilm forming Figure 2. Mean absorbance of Pseudomoas aeruginosa isolates subjected to the biofilm formation test. Analysis of biofilm formation and multidrug resistance (MDR) classification among the isolates, as shown in Table 2, revealed that 37.5% (12/32) of the isolates were both MDR and moderate/strong biofilm producers, which was the same rate observed in non-MDR isolates that were also moderate/strong biofilm producers. Addition- ally, 25% (8/32) of the isolates were weak/non-biofilm producers and non-MDR. No statistically significant relationship was observed between biofilm production and multidrug resistance. Table 2. Biofilm formation and multidrug resistance classification of Pseudomonas aeruginosa isolates from the oral cavity of patients admitted in the ICUs. Oral cavity Biofilm model MDR classification Strong/moderate producer Weak/non producer MDR 37,5% (12/32) 0 Non-MDR 37,5% (12/32) 25% (8/32) Discussion Patients hospitalized in intensive care units (ICUs) are vulnerable to various compli- cations during their stay, including oral health issues. Factors such as malnutrition, the presence of endotracheal tubes, low fluid intake, poor oral hygiene, and others contribute to susceptibility to pathogenic bacterial colonization, which can lead to multiple infections4. In this study, out of 114 oral swabs collected from patients in ICUs across three hos- pitals, 28% (32/114) were colonized with Pseudomonas aeruginosa. A study con- ducted by Bratic et al.23 (2021) found a 14.6% (33/225) rate of oral colonization by P. aeruginosa among ICU patients in Croatia. These findings are alarming, as oral col- 8 Silva et al. Braz J Oral Sci. 2025;24:e257686 onization by Gram-negative bacteria can become a gateway for other types of infec- tions. Furthermore, colonization by P. aeruginosa is concerning due to its prevalence in cases of hospital-acquired pneumonia (HAP), which has been associated with aspi- ration of oral colonization and its multidrug resistance24,25. In this study, 37.5% of the isolates were classified as multidrug-resistant (MDR), with the highest rates of non-susceptibility observed to levofloxacin (46.8%), imipenem (43.7%), and meropenem (40.6%). Levofloxacin is part of the fluoroquinolone class, a major group of antibiotics used for treating P. aeruginosa infections. It is easily administered, being the only therapy given orally, which contributes to its widespread use26. Additionally, the second highest rates of non-susceptibility observed in the study were to carbapenems. Few studies have addressed the colonization of Gram-negative bacilli, specifically P. aeruginosa, in the oral cavity, but some report colonization by this pathogen with non-susceptibility to carbapenems27,28, which is alarming given that this antimicrobial class is one of the last-resort treatments for infections and resistance to it is asso- ciated with adverse clinical outcomes29. Moreover, these strains have various resis- tance mechanisms, such as carbapenemases, which not only confer resistance to carbapenems but also extend to other antimicrobial classes17. In this study, antibiotic resistance mechanisms were investigated using pheno- typic methods, revealing positivity for AmpC and Metallo-β-lactamases. AmpCs are chromosomal enzymes, also known as Pseudomonas-derived cephalosporinases (PDCs), which can induce resistance to β-lactams. While the expression of AmpC alone does not affect carbapenems, it contributes to the resistance phenotype in conjunction with other mechanisms14. This study had limitations regarding the detection of resistance mechanisms, as it was limited to phenotypic detection of enzymes, requiring genotypic information for a complete assessment of the resis- tance profile of P. aeruginosa isolates. Additionally, phenotypic tests have limitations in specificity and sensitivity, particularly concerning false positives and indistinction of enzymes30,31. Of the 32 oral isolates, 75% were moderate/strong biofilm producers. This result is expected, as P. aeruginosa is intrinsically a biofilm producer, considered a virulence factor in the species and associated with horizontal gene transfer of resistance19. Furthermore, the presence of P. aeruginosa in dental plaques has been reported32,33. However, the presence of these species in the oral cavity of ICU patients should be monitored to prevent microaspiration of these pathogens and, consequently, avoid infectious processes such as hospital-acquired pneumonia commonly caused by P. aeruginosa34. In this regard, the process and quality of oral hygiene in patients should be assessed, as they can help prevent the proliferation of these pathogens and reduce infections, mortality, and length of hospital stay6. In conclusion, based on the results presented in this study, monitoring the oral cavity of ICU patients is necessary to track pathogenic microorganisms, reinforce effective oral hygiene guidelines and protocols, and encourage more robust research on the topic to aid in addressing this issue. 9 Silva et al. Braz J Oral Sci. 2025;24:e257686 Acknowledgments To the Experimental Biology Post-graduate Program (PGBIOEXP), Oswaldo Cruz Foun- dation of Rondônia (Fiocruz-RO) and Tropical Medicine Research Center (CEPEM) for their support in carrying out the project. To the entire Laboratory of Microbiology team of Fiocruz-RO for their support in the experiments. We thank the funding institutions such as: National Institute of Epidemiology in the Western Amazon – INCT – EPIAMO, Ministry of Health; Research Program for SUS (PPSUS) and Foundation to Support the Development of Scientific and Technological Actions and Research in the State of Rondônia (FAPERO). Conflict of Interest The authors have no conflict of interest to disclose. Data availability Datasets related to this article will be available upon request to the corresponding author. Author Contribution Marcos Eduardo Passos da Silva: contributed to the laboratory analyzes; data acqui- sition, performed the analysis, interpretation of data for the work, literature search and revised the manuscript. Luccas Manoel de Melo Suica: perfomed the analysis of data for the work and revised the manuscript. Renata Santos Rodrigues and Anjo Gabriel Carvalho: perfomed the analysis of data for the work and revised the manuscript. Izabelly Vitória Gotara Ramos, Nucia Cristiane da Silva Lima, Mayra Gyovana Leite Belém and Rosimar Pires Esquerdo conducted laboratory analyzes. Najla Benevides Matos: conceptualized the project, interpretation of data, revised the manuscript and supervised the project. All authors actively revised and approved the final version of the manuscript. References 1. 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