Analysis of Biofilm Formation and Small Colony Variant Development under Colistin Pressure in Different Clones of Acinetobacter spp

Analysis of Biofilm Formation and Small Colony Variant Development under Colistin Pressure in Different Clones of Acinetobacter spp | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis of Biofilm Formation and Small Colony Variant Development under Colistin Pressure in Different Clones of Acinetobacter spp Larissa Naneti Rosa, Giovanna Costa da Silva, Sophia Mendes Gonçalves, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9094954/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Acinetobacter baumannii is a Gram-negative bacterium frequently associated with healthcare-associated infections (HAIs) and represents a growing public health concern due to its resistance to multiple antimicrobials. A. baumannii is particularly notable for its ability to form biofilms, complex cellular structures adhered to surfaces, and for its clonal diversity, which complicates infection control. Additionally, the formation of small colony variants (SCVs) has been associated with increased virulence and resistance. This study aimed to analyze biofilm formation and SCV occurrence among A. baumannii clones, as well as to investigate the presence of the bap gene, which is associated with biofilm production. Biofilms were developed under static conditions in 6-well plates at 37°C for 24 hours. Results showed that colistin-resistant A. baumannii strains produced significantly higher biofilm cell densities (5.02 × 10¹¹ CFU/mL) compared to susceptible strains (4.9 × 10¹⁰ CFU/mL; p = 0.0173). Moreover, resistant strains exhibited a higher frequency of SCV generation (5.4 × 10⁻²) than susceptible isolates (1 × 10⁻²; p = 0.0226). Three distinct Acinetobacter baumannii and Acinetobacter calcoaceticus–A. baumannii (ACB) complex clones were identified using Random Amplified Polymorphic DNA (RAPD) analysis. Clone 2 produced the highest biofilm cell density under subinhibitory colistin concentration (1.29 × 10¹⁴ CFU/mL), which was significantly greater than that of Clone 1 (5.22 × 10¹¹ CFU/mL; p = 0.033) and Clone 3 (1.79 × 10¹¹ CFU/mL; p = 0.021). Additionally, the bap gene was detected in all isolates, suggesting its important role in adhesion and persistence. In conclusion, the ability of Acinetobacter baumannii to form dense biofilms, together with the emergence of small colony variants (SCVs), represents a major challenge for the effective treatment and control of healthcare-associated infections (HAIs). These adaptive traits contribute to increased persistence in clinical environments, enhanced tolerance to antimicrobial agents, and difficulties in eradication, highlighting the need for improved strategies to prevent and manage these infections. Acinetobacter baumannii SCVs biofilms clonal diversity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Acinetobacter baumannii is a nosocomial, non-fermenting, aerobic, non-motile, catalase-positive, oxidase-negative, and non fastidious Gram-negative coccobacillus [ 1 ]. It grows on commonly used culture media, such as MacConkey agar, blood agar, and tryptic soy agar (TSA) [ 2 ]. Ubiquitous in nature, it has been identified as part of the microbiota of the throat and rectum and has also been isolated from food and body lice [ 3 ]. A. baumannii belongs to the Acinetobacter calcoaceticus-A. baumannii (ACB) complex, which includes primarily environmental species such as A. calcoaceticus and clinically significant species such as A. baumannii , Acinetobacter pittii , Acinetobacter nosocomialis , Acinetobacter seifertii , and Acinetobacter dijkshoorniae . Of these, A. baumannii is the most clinically relevant due to its extensive repertoire of antimicrobial resistance [ 4 ]. Furthermore, it is part of the “ESKAPEE” group, an acronym for Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , Enterobacter spp., and Escherichia coli , which comprises pathogens characterized by multidrug resistance and high virulence and is associated with healthcare-associated infections (HAIs) [ 76 ]. In humans, A. baumannii is considered an opportunistic pathogen, primarily affecting hospitalized patients. It is a leading cause of ventilator-associated pneumonia, bloodstream infections, urinary tract infections, and meningitis [ 5 ]. In the United States, it is estimated that approximately 12,000 A. baumannii infections occur annually, resulting in around 500 deaths [ 6 ]. In Brazil, one study found that 70–80% of A. baumannii isolates were resistant to carbapenems, contributing to an estimated 50,000 to 100,000 deaths caused by the six World Health Organization (WHO) priority pathogens [ 7 , 8 ]. Outbreaks have since been reported on all major continents, with multidrug-resistant (MDR) isolates accounting for up to 44% of infections [ 5 ]. In response to this threat, organizations such as the European Centre for Disease Prevention and Control (ECDC), the Infectious Diseases Society of America (IDSA), the WHO, and the U.S. Centers for Disease Control and Prevention (CDC) have declared MDR A. baumannii a critical global health threat, elevating it from a “serious” to an “urgent” threat in 2019 [ 5 ]. Due to high resistance rates and associated mortality, the WHO has categorized carbapenem-resistant A. baumannii as a critical priority pathogen for monitoring dissemination and for the development of new drugs [ 7 ]. A. baumannii exhibits a wide range of resistance mechanisms, including the production of β-lactamases and modifications targeting fluoroquinolones, aminoglycosides, and polymyxins [ 9 ]. Non enzymatic mechanisms, such as efflux pumps, reduced membrane permeability, and target site modifications, also contribute to its resistance profile. This accumulation of mechanisms, together with its capacity to form biofilms, has significantly limited the effectiveness of available antimicrobial treatments [ 10 ]. Biofilms are structured communities of microorganisms adhered to surfaces and embedded within a matrix of extracellular polymeric substances (EPS), which function as a survival strategy. This matrix not only protects bacteria from environmental stressors but also impedes the penetration and effectiveness of antimicrobial agents [ 11 , 12 , 79 ]. In mucoid strains, biofilm formation tends to be enhanced, with alginic acid as the predominant exopolysaccharide [ 13 ], while non-mucoid strains predominantly produce Psl and Pel polysaccharides and exhibit lower biofilm forming capacity [ 14 ]. Biofilms may consist of a single species or a consortium of different microorganisms [ 15 ]. Their development occurs in defined stages: (i) initial attachment to surfaces; (ii) maturation with EPS production; and (iii) dispersion, in which cells detach and revert to their planktonic state [ 16 ]. Within biofilms, bacteria communicate via quorum sensing (QS), a population density dependent signaling system that regulates gene expression and influences cellular physiology [ 17 ]. In A. baumannii , QS, along with bacterial appendages and surface structures, plays a crucial role in biofilm formation. External factors such as nutrient limitation, pH, cation concentration, and temperature also influence this process [ 18 ]. Among the proteins involved in biofilm formation is Bap, encoded by the bap gene, which mediates intercellular adhesion and biofilm stability [ 19 ]. This protein is highly immunogenic and has been shown to increase antibody production in mice, making it a potential therapeutic target [ 20 ]. Additionally, outer membrane protein A (OmpA) and pili are implicated in biofilm development. The latter are encoded by the csu operon, which facilitates adherence through the formation of pilus-like structures [ 21 , 22 ]. However, the rising incidence of infections, coupled with the widespread misuse of antimicrobials, has contributed to the emergence of carbapenem-resistant A. baumannii (CRAB). In such cases, last-resort treatments include polymyxins and tigecycline [ 23 , 24 ]. Resistance in A. baumannii is often mediated by class A, C, and D β-lactamases and metallo-β-lactamases, in addition to efflux pumps that can alter antimicrobial binding sites and enhance resistance [ 10 , 25 ]. Most MDR or extensively drug-resistant (XDR) A. baumannii isolates belong to two major globally disseminated clones (GC1 and GC2), initially identified in Europe during the 1970s [ 26 ]. To date, at least nine successful global clones (GC1–GC9) have been described [ 27 ]. These clones are often associated with multidrug resistance and have been implicated in numerous hospital outbreaks [ 2 ]. GC2, in particular, appears to be the most prevalent lineage associated with carbapenem resistance [ 28 ]. However, other clonal lineages, including GC4 and GC5, have been reported in Europe and South America [ 27 ]. GC6, first described in Italy in 2006, demonstrated resistance to all tested antimicrobials except colistin [ 27 , 29 ]. GC7 has been predominant in Bolivia and Uruguay, although it has since been detected in other regions [ 30 ]. In contrast, GC8 and GC9 have so far been identified only as single isolates [ 27 , 31 ]. Another important phenotypic characteristic in A. baumannii is the formation of small colony variants (SCVs), which are slow-growing, auxotrophic subpopulations that produce small, translucent colonies and exhibit distinct phenotypic and pathogenic features [ 32 ]. Typically non-pigmented or spot pigmented on blood agar, these colonies often yield negative biochemical test results [ 33 ]. Clinically, SCVs demonstrate increased intracellular persistence and reduced susceptibility to antimicrobials, contributing to chronic or recurrent infections, particularly in patients with cystic fibrosis, osteomyelitis, or infections related to medical devices such as endotracheal tubes and mechanical ventilators [ 34 , 35 , 80 ]. In A. baumannii , SCVs are associated with altered cell morphology, surface motility, enhanced biofilm formation, increased antimicrobial resistance, and elevated virulence [ 32 ]. These features may hinder proper identification in clinical laboratories and lead to treatment failure [ 35 , 36 ]. Given these challenges, this study aims to investigate the clonal diversity, biofilm-forming capacity, and production of small colony variants (SCVs) among clinical isolates of the ACB complex under conditions with and without subinhibitory concentrations of colistin (polymyxin E) used as a stressor. The ultimate goal is to provide robust data that may support the development of more effective therapeutic strategies for affected patients. Materials and Methods Origin of Bacterial Samples, Identification, and Susceptibility Profile of Isolates A total of 42 A. baumannii isolates were analyzed and divided into two groups. Group 1 (n = 24) included isolates obtained from patients admitted to Hospital das Clínicas (HC) of the Universidade Federal de Minas Gerais (UFMG) between March 2012 and March 2013. The isolates were obtained from catheter tips (36%), tracheal aspirates (28%), blood cultures (24%), urine cultures (8%), and surgical wounds (4%). Group 2 (n = 18) consisted of carbapenem-resistant isolates collected in 2019 at Hospital João XXIII, Belo Horizonte, Minas Gerais, with approval from the Research Center of the Hospital Foundation of the State of Minas Gerais – FHEMIG (Process No. 2270.01.0027733/2019-80). Their sources included skin secretions (39.13%), tracheal secretions (30.43%), catheter tips (13.04%), urine (8.69%), blood (4.34%), and cerebrospinal fluid (4.34%). Group 1 isolates were identified by the microbiology department at HC-UFMG and confirmed via PCR at the Laboratory of Cell Biology of Microorganisms, UFMG, through detection of blaOXA-51 and other oxacillinase genes. Group 2 isolates were identified phenotypically using the automated Vitek 2® system and confirmed by MALDI-TOF MS. Group 1 samples were stored at − 80°C in BHI broth with glycerol, and Group 2 samples at − 20°C in nutrient broth with glycerol, at UFMG and the Universidade Federal de São João del-Rei (UFSJ), respectively. Minimum inhibitory concentrations (MICs) for colistin were determined for all isolates at their institutions of origin. In Group 1, the E-test® showed resistance to ampicillin/sulbactam (61.3%), ceftazidime (71%), gentamicin (24.2%), meropenem (98.4%), and tigecycline (48.4%), with all isolates remaining sensitive to polymyxin B using the MIC method. For Group 2, susceptibility testing was performed using the Vitek 2® system and broth microdilution, confirming complete resistance to carbapenems. Antimicrobial concentrations followed Clinical and Laboratory Standards Institute (CLSI) guidelines applicable to the year of collection. Species Confirmation via gyrB Gene PCR All isolates were confirmed at the species level by PCR at the Laboratory of Microorganism Cell Biology (LBCM), Institute of Biological Sciences (ICB), Federal University of Minas Gerais (UFMG), following the protocol described by Higgins et al. (2007). Specific primers targeting the gyrB gene were used: sp4F (5’-CACGCCGTAAGAGTGCATTA), sp4R (5’-AACGGAGCTTGTCAGGGTTA), and sp2F (5’-GTTCCTGATCCGAAATTCTCG), with an annealing temperature of 56°C. Amplification of 294 bp and 490 bp fragments confirmed A. baumannii . The ATCC 19606 strain served as a positive control. DNA was extracted from colonies grown on BHI agar (Neogen Corporation, Michigan) and incubated in BHI broth (Neogen Corporation, Michigan) at 37°C, following a protocol adapted from Seratti and Maniglia (2019). PCR amplification used a Master Mix containing Taq DNA polymerase (Biotechnology and Services, pht, Brazil), dNTPs (NeoTaq, Brazil), MgCl₂, and primers (Biotechnology and Services, pht, Brazil). Products were resolved by 1.5% agarose gel electrophoresis (Life Technologies, Inc., USA), stained with ethidium bromide, and visualized under UV light. Confirmed isolates were used for all subsequent experiments. Genotypic Characterization Random Amplified Polymorphic DNA (RAPD) analysis was conducted using the DAF4 primer with an annealing temperature of 50°C. Each 100 µL reaction included Taq DNA polymerase, dNTPs, MgCl₂, Tris-HCl, KCl, DMSO, and the DAF4 primer. Amplified products were separated by 2% agarose gel electrophoresis [ 37 ]. Clonal relationships were evaluated using the Dice coefficient and PyElph 1.4 software, with an 85% similarity threshold defining clonal identity [ 37 ]. Detection of the bap Gene Associated with Biofilm Formation The bap gene was detected via PCR using primers F (5’-ATGCCTGAGATACAAATTAT) and R (5’-GTCAATCGTAAAGGTAACG) (IDT, USA), with an annealing temperature of 60°C [ 38 ]. Each 25 µL reaction included buffer, Taq DNA polymerase, dNTPs, MgCl₂, primers, and nuclease free water. PCR products were analyzed on 1% agarose gels, stained with ethidium bromide, and visualized under UV light. Minimum Inhibitory Concentration (MIC) Broth Microdilution MIC testing for colistin in Group 1 isolates was performed by broth microdilution according to BrCAST (2024) guidelines. Colistin stock solutions (1 mg/mL) were stored at − 20°C. Inocula (5 × 10⁵ CFU/mL) were prepared by serial dilution and confirmed by plate counting. Testing was performed in 96-well plates with Mueller-Hinton broth supplemented with Tween 0.002% and incubated at 37°C for 24 h. MICs were read visually as the lowest concentration showing no visible growth, with test concentrations ranging from 0.25 to 32 µg/mL. MIC values for Group 2 had been previously determined. Biofilm Formation and SCV Analysis Biofilm formation was assessed by CFU (colony-forming units) quantification, following Campos et al. (2016) with modifications. Overnight cultures in BHI broth at 37°C were serially diluted (10⁻⁸), plated on BHI agar, and incubated to determine CFU/mL. Inocula were adjusted to 1.0 × 10⁶ CFU/mL. Biofilms were grown in 6-well polystyrene plates at 37°C for 24 h. After removal of planktonic cells and washing with 1× PBS, adhered cells were detached by scraping, serially diluted, and plated. CFUs were counted manually and adjusted for dilution factors. To assess SCV formation, biofilm plates were reincubated for 7 days at 37°C. SCVs were evaluated on days 3 and 7 based on phenotype: colonies approximately 10 times smaller than the wild type and visible only after 48 h. Confirmation of SCVs was performed via gyrB gene PCR. For SCV analysis under antimicrobial pressure, biofilms were exposed to subinhibitory colistin concentrations (0.125 µg/mL) for 24 h. Following treatment, cells were detached, diluted, plated, and incubated for 72 h. SCVs were identified and confirmed via gyrB PCR. Statistical Analysis All experiments were conducted with at least three independent biological replicates, including antibiotic susceptibility testing. Statistical analysis was performed using SigmaXL software. Data comparisons were made using the non-parametric Mann Whitney Wilcoxon test, with significance set at p ≤ 0.05. Results and Discussion Confirmation of A. baumannii Species A total of 42 bacterial isolates were recovered from hospitalized patients, and species identification was confirmed via PCR targeting the gyrB gene (Fig. 1). Despite prior classification as A. baumannii , 28.57% (12/42) of the isolates were identified as other species within the ACB complex. The use of gyrB based PCR for Acinetobacter species identification has been well established for over a decade. For instance, Lee et al. (2014) demonstrated its superior accuracy compared to automated methods such as Vitek2 (90.5% vs. 100% concordance with Kraken 2 and ANI). Thus, gyrB targeted PCR remains a reliable and essential tool for epidemiological studies involving clinical Acinetobacter strains [ 39 ]. Of the isolates analyzed, 71.42% (30/42) were confirmed as A. baumannii (Table 1). Differentiating species within the ACB complex is critical but challenging due to their close genetic relationships and phenotypic similarities [ 39 ]. It should be noted that the blaOXA-51-like gene can also be used to identify A. baumannii , as it has been found in virtually every isolate of this species within the ACB complex [ 77 ]. In fact, combined identification methods are often required; therefore, PCR amplification of the gyrB gene and MALDI-TOF mass spectrometry represent optimal approaches for identifying members of the Acinetobacter calcoaceticus-A. baumannii complex [ 60 ]. Clonal Diversity Analysis The spread of resistant bacterial clones in healthcare settings poses a significant threat to public health by complicating treatment and increasing patient morbidity and mortality. Accurate clone identification is critical for infection control, outbreak tracking, and the development of prevention strategies. Molecular biology tools such as PCR facilitate the mapping of genetic diversity and resistance profiles, aiding personalized treatment and health policy development [ 38 ]. Of the 42 Acinetobacter spp. isolates analyzed, 23 (55%) A. baumannii strains were selected as a representative subset for clonal profiling, ensuring coverage of variation in both clinical origin and colistin resistance and susceptibility. This subset included 3 isolates from tracheal secretions, 4 from tracheal aspirates, 3 from urine, 5 from catheter tip samples, 5 from blood cultures, and 3 from skin secretions. Of these, 14 were colistin-resistant and 9 were colistin-sensitive. The selection of these isolates was not based solely on colistin resistance but rather on their identification as A. baumannii and the need to represent the diversity of clinical sources and epidemiological characteristics within the collection. Two A. baumannii clones were identified in clusters 1 (12 strains) and 2 (7 strains), with isolates exhibiting both colistin-sensitive and colistin-resistant profiles (Fig. 2). Cluster 3 (4 strains) consisted of species belonging to the ACB complex. The dominant clones were associated with HC-UFMG (Group 1) and Hospital João XXIII, both located in Belo Horizonte, Minas Gerais (Group 2). Clone identification is crucial for understanding hospital infection dynamics and selecting appropriate antimicrobial therapies. The limited number of clones identified may reflect a recent population contraction of A. baumannii , possibly due to its narrow ecological niche. Unlike other Acinetobacter species with broad environmental distribution, A. baumannii is primarily associated with human infections, which may restrict its genetic diversity [ 40 ]. Over the past three decades, A. baumannii has gained clinical relevance due to its capacity to acquire and express antimicrobial resistance genes. Strains belonging to the ACB complex (Groups 1 and 2) in cluster 3 included both sensitive and colistin-resistant isolates. The observed clonal diversity can be attributed to antimicrobial pressure, adaptation to hospital environments, and efficient transmission among patients and surfaces. This pathogen is frequently associated with nosocomial infections, particularly in intensive care units (ICUs), where antibiotic misuse and failures in infection control enhance its persistence [ 40 ]. Clone 1 isolates were predominantly obtained from tracheal secretions (bacterial pneumonia) and catheter tips (bloodstream infections), whereas clone 2 isolates were mainly recovered from tracheal aspirates (bacterial pneumonia) and blood cultures (bloodstream infections) (Fig. 2). Most A. baumannii bloodstream infections are associated with medical care [ 61 ], while bacterial pneumonia caused by A. baumannii is linked to high mortality and morbidity, especially in critically ill ICU patients [ 62 ]. The data from this study indicate that both clones 1 and 2 are involved in these infections, suggesting that they may share similar virulence factors that enable colonization and infection of these sites. When considering colistin resistance, clone 2 comprised isolates with higher resistance rates (6 R / 1 S) compared to clone 1 (5 R / 7 S) (p = 0.05) (Fig. 2). The detection of clone 2, characterized by a higher proportion of resistant isolates, underscores the importance of monitoring infections in hospital settings. Previous studies have identified the ST2 global clone as the predominant CRAB lineage in ICUs [ 63 ]. Colistin Minimum Inhibitory Concentration (MIC) Colistin susceptibility was assessed via broth microdilution following BrCAST (2024) criteria (Table 1). A. baumannii is commonly associated with severe infections in immunosuppressed ICU patients [ 41 ]. In this study, isolates from tracheal aspirates, catheter tips, and blood cultures showed high levels of colistin resistance (26 resistant strains; MIC > 4 µg/mL). Although polymyxins (e.g., colistin) serve as last-line treatments, their overuse has contributed to the emergence of resistance, posing a major public health concern [ 42 ]. In this study, 61.90% (26/42) of isolates were resistant to colistin, with Group 1 showing 75% resistance compared to 38.9% in Group 2. MIC values ranged from 4 to 32 µg/mL. Approximately 70% of isolates from bacterial pneumonia (7/10) and 64.0% of bloodstream infection isolates (9/14) were resistant to colistin (Table 1). Among colistin-resistant A. baumannii isolates, Group 1 had a higher number of resistant strains (15/18) than Group 2 (3/12) (p = 0.0024). Colistin-resistant A. baumannii strains are often associated with pneumonia and bloodstream infections and represent a major cause of nosocomial infections with significant morbidity and mortality. This pattern is consistent with previous reports of CRAB infections, in which mortality rates particularly among patients with pneumonia and bloodstream infections may reach approximately 60% [ 71 , 72 ]. Biofilm Cell Density Quantification In this study, significant differences in biofilm cell density were observed between colistin-resistant and colistin-sensitive A. baumannii strains. Resistant strains formed denser biofilms (5.02 × 10¹¹ CFU/mL) than sensitive strains (4.9 × 10¹⁰ CFU/mL; p = 0.0173) (Graph 1A). This difference may be related to mechanisms such as antimicrobial defense [ 43 , 44 ], metabolic adaptation [ 45 ], increased structural protection [ 16 ], and interspecies interactions [ 46 ]. The combination of antimicrobial resistance and biofilm formation makes A. baumannii a formidable pathogen in healthcare settings, where it can cause pneumonia, bloodstream infections, wound infections, and urinary tract infections [ 64 ]. Biofilms provide a protective environment for bacterial cells and further enhance their resistance to antimicrobial agents; they are also associated with persistent infections and the development of chronic disease. Within the ACB complex, no significant differences in biofilm density were observed between colistin-resistant (1.92 × 10¹⁰ CFU/mL) and sensitive strains (1.25 × 10¹¹ CFU/mL; p ≥ 0.1). Factors such as surface adhesion [ 47 ], nutrient availability [ 48 ], humidity [ 49 ], physicochemical conditions, and quorum sensing [ 50 ] influence biofilm development. Previous studies (Kaplan, 2011; Sato et al., 2018; Wang et al., 2010) have shown that subinhibitory antibiotic concentrations can stimulate biofilm formation and increase bacterial virulence. Consistent with these findings, sensitive A. baumannii strains exposed to subinhibitory colistin concentrations produced significantly denser biofilms (4.6 × 10¹¹ CFU/mL) than unexposed strains (4.9 × 10¹⁰ CFU/mL; p = 0.0136) (Graph 1B). Among the identified clones, clone 2 exhibited the highest biofilm density under antimicrobial pressure, followed by clones 1 and 3 (Graph 1C). The enhanced biofilm-forming capacity of clone 2 under colistin pressure (1.29 × 10¹⁴ CFU/mL) suggests increased virulence and persistence, representing a significant public health concern. Isolates with higher biofilm production were mainly obtained from tracheal aspirates and tracheal secretions (bacterial pneumonia) (Graph 1D). A. baumannii exhibits increased biofilm formation at the solid liquid interface, as observed in pneumonia cases. Previous studies have shown that isolates from patients with aspiration pneumonia produce substantial amounts of biofilm [ 65 ]. Detection of the bap Gene The bap gene, an important virulence factor in A. baumannii , was screened in all isolates to evaluate its association with biofilm formation (Fig. 4). All A. baumannii and ACB complex isolates carried the bap gene, indicating a 100% prevalence among hospital isolates. In another study, the prevalence of bap was 79.2%, and a significant correlation was observed between antibiotic resistance, biofilm formation, and biofilm-associated genes [ 68 ]. The detection of bap in all clinical specimens analyzed suggests that this gene contributes to biofilm formation, persistence in hospital environments and on medical devices, and the pathogenesis of healthcare associated infections. Its presence was confirmed in both antibiotic-resistant and antibiotic-sensitive isolates. This gene promotes adhesion and colonization in hospital environments, thereby enhancing persistence and resistance to disinfectants and host immune responses [ 57 , 69 ]. Understanding the role of bap is crucial for developing new therapeutic strategies, as previous studies have demonstrated that bap expression leads to robust biofilm formation and increased resistance to antimicrobial treatments [ 58 ]. According to De Gregorio et al. (2015), Bap is highly polymorphic, and this variability may influence biofilm formation in Gram-negative bacteria [ 74 ]. Further molecular studies investigating Bap and its interaction with other proteins may help clarify the differences in biofilm-forming capacity observed between A. baumannii and ACB complex isolates. SCV Formation Under Colistin Pressure Small colony variants (SCVs) are slow-growing bacterial subpopulations characterized by altered metabolism and increased persistence [ 52 , 53 ]. Their formation was evaluated in both A. baumannii and ACB complex isolates, with and without exposure to subinhibitory concentrations of colistin (Graph 1E and 1F). Colistin-resistant A. baumannii strains produced more SCVs without antibiotic pressure (5.4 × 10⁻² CFU/mL) than with subinhibitory colistin (1.2 × 10⁻² CFU/mL; p = 0.025). Conversely, colistin-sensitive strains produced more SCVs under subinhibitory colistin exposure (5 × 10⁻² CFU/mL) than without exposure (1 × 10⁻² CFU/mL; p = 0.035). These results suggest that subinhibitory concentrations of colistin can promote biofilm formation in sensitive strains and stimulate the emergence of SCVs, highlighting the potential risks associated with subtherapeutic antimicrobial exposure. These findings are consistent with previous studies showing that subinhibitory antibiotic concentrations can induce the SOS response and increase SCV formation [ 54 ]. The frequency of SCV generation was significantly higher in colistin-resistant A. baumannii strains (5.4 × 10⁻²) compared to susceptible strains (1 × 10⁻²) (p = 0.0226). Previous studies have shown that phase variation in A. baumannii contributes to resistance against several antibiotics, including variations in colistin MIC values [ 66 ]. SCVs are particularly concerning because they require 48–72 hours to grow and are often undetectable by conventional microbiological methods, representing a significant public health concern. Their increased antibiotic resistance allows them to persist in environments with high antimicrobial pressure, such as hospitals, contributing to infections that are difficult to treat [ 78 ]. SCV Species Confirmation by gyrB PCR A. baumannii SCVs exhibited altered band patterns compared to their parental strains (Fig. 5). Previous studies have shown that colony phase variation in A. baumannii the process that generates opaque (virulent) and translucent (avirulent) colonies is regulated by DNA rearrangements involving recombinase machinery and DNA methylation-associated epigenetic modifications [ 70 ]. The formation of SCVs in A. baumannii may involve alterations in the gyrB gene sequence as well as DNA rearrangements mediated by recombinase activity and epigenetic modifications linked to DNA methylation. The gyrB gene, together with gyrA, encodes DNA gyrase, which plays a crucial role in DNA replication, transcription, and recombination [ 75 ]. Therefore, the altered band patterns observed in the gyrB gene of SCVs are likely due to sequence variations and should be further investigated to clarify the molecular mechanisms underlying SCV formation. Conclusion This study demonstrated that colistin-resistant strains of A. baumannii produce significantly higher biofilm cell densities and exhibit increased formation of SCVs, both of which are associated with enhanced resistance and persistence in hostile environments. In conclusion, the ability of A. baumannii to form dense biofilms, combined with SCVs formation and clonal diversity, represents a significant challenge in the treatment of HAIs. The global dissemination of high risk clones underscores the urgent need for effective prevention and control strategies. These findings reinforce the need for robust infection control measures, antimicrobial stewardship, and the development of targeted therapies to mitigate the threat posed by colistin resistant A. baumannii in healthcare environments. Furthermore, intensified research into resistance mechanisms, novel therapeutic approaches, and strict surveillance measures in healthcare settings is essential. Declarations Conflict of Interest The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. Funding Information UFMG Support Foundation (FUNDEP) Grant Number: 30201*52. Author Contribution Larissa Naneti Rosa, Magna Cristina de Paiva, and Santiago Lattar drafted the main manuscript text. Giovanna Costa da Silva and Sophia Mendes prepared Figures 1–5 and Graph 1. All authors contributed to the revision of the manuscript and approved the final version. 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Brazilian J microbiology: [publication Brazilian Soc Microbiology] 55(4):3921–3932. https://doi.org/10.1007/s42770-024-01550-4 Tuchscherr L, Bischoff M, Lattar SM, Noto Llana M, Pförtner H, Niemann S, Geraci J, Van de Vyver H, Fraunholz MJ, Cheung AL, Herrmann M, Völker U, Sordelli DO, Peters G, Löffler B (2015) Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections. PLoS Pathog 11(4):e1004870. https://doi.org/10.1371/journal.ppat.1004870 Tables Table 1 is available in the Supplementary Files section. Graph 1 Graph 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table030925.pptx GA1.png Graph 1. A) Quantification of biofilm cell density in A. baumannii and ACB complex isolates. B) Quantification of biofilm cell density in A. baumannii and ACB complex isolates under subinhibitory concentrations of colistin. C) Biofilm formation by Acinetobacter Clones under subinhibitory colistin concentrations. D) Biofilm production in A. baumannii isolated from different areas. E) SCV Formation Frequency in A. baumannii and ACB Complex Isolates Exposed or Unexposed to Subinhibitory Colistin. F) Prevalence of SCV Formation in A. baumannii and the ACB Complex. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9094954","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":606369015,"identity":"41a3de7e-487d-419b-96fb-c79363f31e89","order_by":0,"name":"Larissa Naneti Rosa","email":"","orcid":"","institution":"Universidade Federal de Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Larissa","middleName":"Naneti","lastName":"Rosa","suffix":""},{"id":606369016,"identity":"32ae6b0e-5dbd-43e0-b235-972d424fbfa1","order_by":1,"name":"Giovanna Costa da Silva","email":"","orcid":"","institution":"Universidade Federal de Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Giovanna","middleName":"Costa da","lastName":"Silva","suffix":""},{"id":606369017,"identity":"abdb918a-d180-4288-b4ec-deb933746ab8","order_by":2,"name":"Sophia Mendes Gonçalves","email":"","orcid":"","institution":"Universidade Federal de Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Sophia","middleName":"Mendes","lastName":"Gonçalves","suffix":""},{"id":606369018,"identity":"0043bb6e-f88e-4fd3-8a86-afb06cc326fd","order_by":3,"name":"Magna Cristina de Paiva","email":"","orcid":"","institution":"Federal University of São João del-Rei","correspondingAuthor":false,"prefix":"","firstName":"Magna","middleName":"Cristina","lastName":"de Paiva","suffix":""},{"id":606369019,"identity":"c626afe9-9cb8-4771-829e-11495f081541","order_by":4,"name":"Santiago Martin Lattar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYDACdgglx8B8gIGBsYEYLcxAdUDFxgxsCSRqSWwgWot8M/Pzxx9q7qVvOMad/IJxxz3CWgwOsxk2HDhWnLvhGO82C8YzxURoYWYAamFLyN1wv3ebAWNbAjEOY//YcOBfQroB0BbitDAc5jFsONiWkADUsvkBUVoMDvMUzjjbl2A4E2gLQ+IZYhzW3r7hQ8W3BHk+oC0fPu4gxmFIgE2CRA3AWP1Aqo5RMApGwSgYGQAAeEA/RmdiJEoAAAAASUVORK5CYII=","orcid":"","institution":"Universidade Federal de Minas Gerais","correspondingAuthor":true,"prefix":"","firstName":"Santiago","middleName":"Martin","lastName":"Lattar","suffix":""}],"badges":[],"createdAt":"2026-03-11 13:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9094954/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9094954/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104749436,"identity":"cc82c2ef-8062-4b75-aa64-3d344625fca5","added_by":"auto","created_at":"2026-03-16 19:00:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":31469,"visible":true,"origin":"","legend":"\u003cp\u003eBanding pattern of \u003cem\u003eA. baumannii \u003c/em\u003eand other species of theACB complex ona 1.5% agarose gel. PCR amplification usingprimers targeting the gyrBgene (Higgins et al., 2007) shows a 294-bp band common to all \u003cem\u003eAcinetobacter\u003c/em\u003e species, anda distinct490-bp band specific to \u003cem\u003eA. baumannii\u003c/em\u003e. KP: molecular weight marker; CN: negative control; CP: positive control; AC24, AC32, AC37, AC39, AC49, AC55: \u003cem\u003eA. baumannii \u003c/em\u003eisolates; AC33, AC39, AC54: ACB complex isolates.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9094954/v1/f7e7e78ed1532b2b9f6f52b0.png"},{"id":104783448,"identity":"65c231b1-cddf-4cbd-937a-e404f976e2ab","added_by":"auto","created_at":"2026-03-17 07:59:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199348,"visible":true,"origin":"","legend":"\u003cp\u003eDNA fingerprinting pattern of \u003cem\u003eAcinetobacter\u003c/em\u003eisolates obtained using the DAF4 primer, visualized on a 2% agarose gel.\u003c/p\u003e\n\u003cp\u003eR:(colistin resistant); S:(colistin sensitive).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9094954/v1/0a91bf3c3062edeb25f0473a.png"},{"id":104749437,"identity":"a3e48e4a-2ebe-4885-9b2d-57b1c1c216e7","added_by":"auto","created_at":"2026-03-16 19:00:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":950558,"visible":true,"origin":"","legend":"\u003cp\u003eA Microscopic comparison of colony size between a typical \u003cem\u003eA. baumannii \u003c/em\u003ecolony and a \u003cem\u003esmall colony variant \u003c/em\u003e(\u003cem\u003eSCVs\u003c/em\u003e). B \u003cem\u003eSmall colony variants \u003c/em\u003e(\u003cem\u003eSCVs\u003c/em\u003e) of \u003cem\u003eA. baumannii \u003c/em\u003egrown on BHI agar after 3 days of incubation at 37°C\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9094954/v1/f5b1379dda1e2a5edc94fb31.png"},{"id":104783331,"identity":"3a00195a-c99c-4f36-b95f-edf68a1933a8","added_by":"auto","created_at":"2026-03-17 07:58:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14779,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of the \u003cem\u003ebap gene \u003c/em\u003ein isolates of \u003cem\u003eA. baumannii \u003c/em\u003eand species of the ACB complex. PCR amplification using primers F and R shows a 1500-bp band corresponding to the bap gene. Bands were visualized on a 1% agarose gel. MW: molecular weight marker; clinical isolates: AC46, AC40, AC45, A5, B6, C8, J18, J19. ATCC 19606.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9094954/v1/8ae55a9729dea9fa416d0928.jpg"},{"id":104749441,"identity":"d962def4-525e-43cd-bcdc-7c57b80e3bcc","added_by":"auto","created_at":"2026-03-16 19:00:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28290,"visible":true,"origin":"","legend":"\u003cp\u003eBanding pattern of SCVs from \u003cem\u003eA. baumannii \u003c/em\u003eand species of theACB complex ona 1.5% agarose gel. PCR targeting the \u003cem\u003egyrB gene \u003c/em\u003e(Higgins et al., 2007) revealsa 294-bp band common to all \u003cem\u003eAcinetobacter species \u003c/em\u003eanda 490-bp band specific to \u003cem\u003eA. baumannii\u003c/em\u003e. KP: molecular weight marker; CN: negative control; CP: positive control; clinical isolates: B6, K21, L25, N27, AC35.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9094954/v1/96ff468c200bfb14451f32e9.png"},{"id":105668949,"identity":"2c061819-2b6c-4550-a7f5-12cd20eddb0c","added_by":"auto","created_at":"2026-03-29 12:40:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2277576,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9094954/v1/e40b5352-9cb4-414d-a67d-287fa15465a9.pdf"},{"id":104783016,"identity":"e82102c1-03bc-477b-8a4a-9e1d7774c406","added_by":"auto","created_at":"2026-03-17 07:58:06","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":52969,"visible":true,"origin":"","legend":"","description":"","filename":"Table030925.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9094954/v1/c74d5392cdd7cf1cfe3d5933.pptx"},{"id":104749440,"identity":"4b8c97d2-6eef-48c0-8b34-838d9a2de812","added_by":"auto","created_at":"2026-03-16 19:00:40","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":156884,"visible":true,"origin":"","legend":"\u003cp\u003eGraph 1. A) Quantification of biofilm cell density in \u003cem\u003eA. baumannii \u003c/em\u003eand ACB complex isolates. B) Quantification of biofilm cell density in \u003cem\u003eA. baumannii \u003c/em\u003eand ACB complex isolates under subinhibitory concentrations of colistin.\u003c/p\u003e\n\u003cp\u003eC) Biofilm formation by \u003cem\u003eAcinetobacter\u003c/em\u003eClones under subinhibitory colistin concentrations. D) Biofilm production in \u003cem\u003eA. baumannii \u003c/em\u003eisolated from different areas. E) SCV Formation Frequency in \u003cem\u003eA. baumannii\u003c/em\u003eand ACB Complex\u003c/p\u003e\n\u003cp\u003eIsolates Exposed or Unexposed to Subinhibitory Colistin. F) Prevalence of SCV Formation in \u003cem\u003eA. baumannii\u003c/em\u003e and the ACB Complex.\u003c/p\u003e","description":"","filename":"GA1.png","url":"https://assets-eu.researchsquare.com/files/rs-9094954/v1/ce8462bedf5c4c214af00c55.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analysis of Biofilm Formation and Small Colony Variant Development under Colistin Pressure in Different Clones of Acinetobacter spp","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e is a nosocomial, non-fermenting, aerobic, non-motile, catalase-positive, oxidase-negative, and non fastidious Gram-negative coccobacillus [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It grows on commonly used culture media, such as MacConkey agar, blood agar, and tryptic soy agar (TSA) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Ubiquitous in nature, it has been identified as part of the microbiota of the throat and rectum and has also been isolated from food and body lice [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eA. baumannii\u003c/em\u003e belongs to the \u003cem\u003eAcinetobacter calcoaceticus-A. baumannii\u003c/em\u003e (ACB) complex, which includes primarily environmental species such as \u003cem\u003eA. calcoaceticus\u003c/em\u003e and clinically significant species such as \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eAcinetobacter pittii\u003c/em\u003e, \u003cem\u003eAcinetobacter nosocomialis\u003c/em\u003e, \u003cem\u003eAcinetobacter seifertii\u003c/em\u003e, and \u003cem\u003eAcinetobacter dijkshoorniae\u003c/em\u003e. Of these, \u003cem\u003eA. baumannii\u003c/em\u003e is the most clinically relevant due to its extensive repertoire of antimicrobial resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, it is part of the \u0026ldquo;ESKAPEE\u0026rdquo; group, an acronym for \u003cem\u003eEnterococcus faecium\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eEnterobacter\u003c/em\u003e spp., and \u003cem\u003eEscherichia coli\u003c/em\u003e, which comprises pathogens characterized by multidrug resistance and high virulence and is associated with healthcare-associated infections (HAIs) [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn humans, \u003cem\u003eA. baumannii\u003c/em\u003e is considered an opportunistic pathogen, primarily affecting hospitalized patients. It is a leading cause of ventilator-associated pneumonia, bloodstream infections, urinary tract infections, and meningitis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the United States, it is estimated that approximately 12,000 \u003cem\u003eA. baumannii\u003c/em\u003e infections occur annually, resulting in around 500 deaths [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In Brazil, one study found that 70\u0026ndash;80% of \u003cem\u003eA. baumannii\u003c/em\u003e isolates were resistant to carbapenems, contributing to an estimated 50,000 to 100,000 deaths caused by the six World Health Organization (WHO) priority pathogens [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Outbreaks have since been reported on all major continents, with multidrug-resistant (MDR) isolates accounting for up to 44% of infections [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In response to this threat, organizations such as the European Centre for Disease Prevention and Control (ECDC), the Infectious Diseases Society of America (IDSA), the WHO, and the U.S. Centers for Disease Control and Prevention (CDC) have declared MDR \u003cem\u003eA. baumannii\u003c/em\u003e a critical global health threat, elevating it from a \u0026ldquo;serious\u0026rdquo; to an \u0026ldquo;urgent\u0026rdquo; threat in 2019 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Due to high resistance rates and associated mortality, the WHO has categorized carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e as a critical priority pathogen for monitoring dissemination and for the development of new drugs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e exhibits a wide range of resistance mechanisms, including the production of β-lactamases and modifications targeting fluoroquinolones, aminoglycosides, and polymyxins [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Non enzymatic mechanisms, such as efflux pumps, reduced membrane permeability, and target site modifications, also contribute to its resistance profile. This accumulation of mechanisms, together with its capacity to form biofilms, has significantly limited the effectiveness of available antimicrobial treatments [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiofilms are structured communities of microorganisms adhered to surfaces and embedded within a matrix of extracellular polymeric substances (EPS), which function as a survival strategy. This matrix not only protects bacteria from environmental stressors but also impedes the penetration and effectiveness of antimicrobial agents [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. In mucoid strains, biofilm formation tends to be enhanced, with alginic acid as the predominant exopolysaccharide [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], while non-mucoid strains predominantly produce Psl and Pel polysaccharides and exhibit lower biofilm forming capacity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiofilms may consist of a single species or a consortium of different microorganisms [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Their development occurs in defined stages: (i) initial attachment to surfaces; (ii) maturation with EPS production; and (iii) dispersion, in which cells detach and revert to their planktonic state [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Within biofilms, bacteria communicate via quorum sensing (QS), a population density dependent signaling system that regulates gene expression and influences cellular physiology [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In \u003cem\u003eA. baumannii\u003c/em\u003e, QS, along with bacterial appendages and surface structures, plays a crucial role in biofilm formation. External factors such as nutrient limitation, pH, cation concentration, and temperature also influence this process [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the proteins involved in biofilm formation is Bap, encoded by the \u003cb\u003ebap\u003c/b\u003e gene, which mediates intercellular adhesion and biofilm stability [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This protein is highly immunogenic and has been shown to increase antibody production in mice, making it a potential therapeutic target [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, outer membrane protein A (OmpA) and pili are implicated in biofilm development. The latter are encoded by the \u003cb\u003ecsu\u003c/b\u003e operon, which facilitates adherence through the formation of pilus-like structures [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, the rising incidence of infections, coupled with the widespread misuse of antimicrobials, has contributed to the emergence of carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e (CRAB). In such cases, last-resort treatments include polymyxins and tigecycline [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Resistance in \u003cem\u003eA. baumannii\u003c/em\u003e is often mediated by class A, C, and D β-lactamases and metallo-β-lactamases, in addition to efflux pumps that can alter antimicrobial binding sites and enhance resistance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMost MDR or extensively drug-resistant (XDR) \u003cem\u003eA. baumannii\u003c/em\u003e isolates belong to two major globally disseminated clones (GC1 and GC2), initially identified in Europe during the 1970s [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To date, at least nine successful global clones (GC1\u0026ndash;GC9) have been described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These clones are often associated with multidrug resistance and have been implicated in numerous hospital outbreaks [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. GC2, in particular, appears to be the most prevalent lineage associated with carbapenem resistance [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, other clonal lineages, including GC4 and GC5, have been reported in Europe and South America [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. GC6, first described in Italy in 2006, demonstrated resistance to all tested antimicrobials except colistin [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. GC7 has been predominant in Bolivia and Uruguay, although it has since been detected in other regions [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In contrast, GC8 and GC9 have so far been identified only as single isolates [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother important phenotypic characteristic in \u003cem\u003eA. baumannii\u003c/em\u003e is the formation of small colony variants (SCVs), which are slow-growing, auxotrophic subpopulations that produce small, translucent colonies and exhibit distinct phenotypic and pathogenic features [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Typically non-pigmented or spot pigmented on blood agar, these colonies often yield negative biochemical test results [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Clinically, SCVs demonstrate increased intracellular persistence and reduced susceptibility to antimicrobials, contributing to chronic or recurrent infections, particularly in patients with cystic fibrosis, osteomyelitis, or infections related to medical devices such as endotracheal tubes and mechanical ventilators [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. In \u003cem\u003eA. baumannii\u003c/em\u003e, SCVs are associated with altered cell morphology, surface motility, enhanced biofilm formation, increased antimicrobial resistance, and elevated virulence [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These features may hinder proper identification in clinical laboratories and lead to treatment failure [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven these challenges, this study aims to investigate the clonal diversity, biofilm-forming capacity, and production of small colony variants (SCVs) among clinical isolates of the ACB complex under conditions with and without subinhibitory concentrations of colistin (polymyxin E) used as a stressor. The ultimate goal is to provide robust data that may support the development of more effective therapeutic strategies for affected patients.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eOrigin of Bacterial Samples, Identification, and Susceptibility Profile of Isolates\u003c/p\u003e \u003cp\u003eA total of 42 \u003cem\u003eA. baumannii\u003c/em\u003e isolates were analyzed and divided into two groups. Group 1 (n\u0026thinsp;=\u0026thinsp;24) included isolates obtained from patients admitted to Hospital das Cl\u0026iacute;nicas (HC) of the Universidade Federal de Minas Gerais (UFMG) between March 2012 and March 2013. The isolates were obtained from catheter tips (36%), tracheal aspirates (28%), blood cultures (24%), urine cultures (8%), and surgical wounds (4%). Group 2 (n\u0026thinsp;=\u0026thinsp;18) consisted of carbapenem-resistant isolates collected in 2019 at Hospital Jo\u0026atilde;o XXIII, Belo Horizonte, Minas Gerais, with approval from the Research Center of the Hospital Foundation of the State of Minas Gerais \u0026ndash; FHEMIG (Process No. 2270.01.0027733/2019-80). Their sources included skin secretions (39.13%), tracheal secretions (30.43%), catheter tips (13.04%), urine (8.69%), blood (4.34%), and cerebrospinal fluid (4.34%).\u003c/p\u003e \u003cp\u003eGroup 1 isolates were identified by the microbiology department at HC-UFMG and confirmed via PCR at the Laboratory of Cell Biology of Microorganisms, UFMG, through detection of blaOXA-51 and other oxacillinase genes. Group 2 isolates were identified phenotypically using the automated Vitek 2\u0026reg; system and confirmed by MALDI-TOF MS. Group 1 samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in BHI broth with glycerol, and Group 2 samples at \u0026minus;\u0026thinsp;20\u0026deg;C in nutrient broth with glycerol, at UFMG and the Universidade Federal de S\u0026atilde;o Jo\u0026atilde;o del-Rei (UFSJ), respectively.\u003c/p\u003e \u003cp\u003eMinimum inhibitory concentrations (MICs) for colistin were determined for all isolates at their institutions of origin. In Group 1, the E-test\u0026reg; showed resistance to ampicillin/sulbactam (61.3%), ceftazidime (71%), gentamicin (24.2%), meropenem (98.4%), and tigecycline (48.4%), with all isolates remaining sensitive to polymyxin B using the MIC method. For Group 2, susceptibility testing was performed using the Vitek 2\u0026reg; system and broth microdilution, confirming complete resistance to carbapenems. Antimicrobial concentrations followed Clinical and Laboratory Standards Institute (CLSI) guidelines applicable to the year of collection.\u003c/p\u003e \u003cp\u003eSpecies Confirmation via gyrB Gene PCR\u003c/p\u003e \u003cp\u003eAll isolates were confirmed at the species level by PCR at the Laboratory of Microorganism Cell Biology (LBCM), Institute of Biological Sciences (ICB), Federal University of Minas Gerais (UFMG), following the protocol described by Higgins et al. (2007). Specific primers targeting the gyrB gene were used: sp4F (5\u0026rsquo;-CACGCCGTAAGAGTGCATTA), sp4R (5\u0026rsquo;-AACGGAGCTTGTCAGGGTTA), and sp2F (5\u0026rsquo;-GTTCCTGATCCGAAATTCTCG), with an annealing temperature of 56\u0026deg;C. Amplification of 294 bp and 490 bp fragments confirmed \u003cem\u003eA. baumannii\u003c/em\u003e. The ATCC 19606 strain served as a positive control. DNA was extracted from colonies grown on BHI agar (Neogen Corporation, Michigan) and incubated in BHI broth (Neogen Corporation, Michigan) at 37\u0026deg;C, following a protocol adapted from Seratti and Maniglia (2019). PCR amplification used a Master Mix containing Taq DNA polymerase (Biotechnology and Services, pht, Brazil), dNTPs (NeoTaq, Brazil), MgCl₂, and primers (Biotechnology and Services, pht, Brazil). Products were resolved by 1.5% agarose gel electrophoresis (Life Technologies, Inc., USA), stained with ethidium bromide, and visualized under UV light. Confirmed isolates were used for all subsequent experiments.\u003c/p\u003e \u003cp\u003eGenotypic Characterization\u003c/p\u003e \u003cp\u003eRandom Amplified Polymorphic DNA (RAPD) analysis was conducted using the DAF4 primer with an annealing temperature of 50\u0026deg;C. Each 100 \u0026micro;L reaction included Taq DNA polymerase, dNTPs, MgCl₂, Tris-HCl, KCl, DMSO, and the DAF4 primer. Amplified products were separated by 2% agarose gel electrophoresis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Clonal relationships were evaluated using the Dice coefficient and PyElph 1.4 software, with an 85% similarity threshold defining clonal identity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDetection of the bap Gene Associated with Biofilm Formation\u003c/p\u003e \u003cp\u003eThe bap gene was detected via PCR using primers F (5\u0026rsquo;-ATGCCTGAGATACAAATTAT) and R (5\u0026rsquo;-GTCAATCGTAAAGGTAACG) (IDT, USA), with an annealing temperature of 60\u0026deg;C [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Each 25 \u0026micro;L reaction included buffer, Taq DNA polymerase, dNTPs, MgCl₂, primers, and nuclease free water. PCR products were analyzed on 1% agarose gels, stained with ethidium bromide, and visualized under UV light.\u003c/p\u003e \u003cp\u003eMinimum Inhibitory Concentration (MIC) Broth Microdilution\u003c/p\u003e \u003cp\u003eMIC testing for colistin in Group 1 isolates was performed by broth microdilution according to BrCAST (2024) guidelines. Colistin stock solutions (1 mg/mL) were stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Inocula (5 \u0026times; 10⁵ CFU/mL) were prepared by serial dilution and confirmed by plate counting. Testing was performed in 96-well plates with Mueller-Hinton broth supplemented with Tween 0.002% and incubated at 37\u0026deg;C for 24 h. MICs were read visually as the lowest concentration showing no visible growth, with test concentrations ranging from 0.25 to 32 \u0026micro;g/mL. MIC values for Group 2 had been previously determined.\u003c/p\u003e \u003cp\u003eBiofilm Formation and SCV Analysis\u003c/p\u003e \u003cp\u003eBiofilm formation was assessed by CFU (colony-forming units) quantification, following Campos et al. (2016) with modifications. Overnight cultures in BHI broth at 37\u0026deg;C were serially diluted (10⁻⁸), plated on BHI agar, and incubated to determine CFU/mL. Inocula were adjusted to 1.0 \u0026times; 10⁶ CFU/mL. Biofilms were grown in 6-well polystyrene plates at 37\u0026deg;C for 24 h. After removal of planktonic cells and washing with 1\u0026times; PBS, adhered cells were detached by scraping, serially diluted, and plated. CFUs were counted manually and adjusted for dilution factors.\u003c/p\u003e \u003cp\u003eTo assess SCV formation, biofilm plates were reincubated for 7 days at 37\u0026deg;C. SCVs were evaluated on days 3 and 7 based on phenotype: colonies approximately 10 times smaller than the wild type and visible only after 48 h. Confirmation of SCVs was performed via gyrB gene PCR. For SCV analysis under antimicrobial pressure, biofilms were exposed to subinhibitory colistin concentrations (0.125 \u0026micro;g/mL) for 24 h. Following treatment, cells were detached, diluted, plated, and incubated for 72 h. SCVs were identified and confirmed via gyrB PCR.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted with at least three independent biological replicates, including antibiotic susceptibility testing. Statistical analysis was performed using SigmaXL software. Data comparisons were made using the non-parametric Mann Whitney Wilcoxon test, with significance set at p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eConfirmation of \u003cem\u003eA. baumannii\u003c/em\u003e Species\u003c/p\u003e \u003cp\u003eA total of 42 bacterial isolates were recovered from hospitalized patients, and species identification was confirmed via PCR targeting the gyrB gene (Fig.\u0026nbsp;1). Despite prior classification as \u003cem\u003eA. baumannii\u003c/em\u003e, 28.57% (12/42) of the isolates were identified as other species within the ACB complex. The use of gyrB based PCR for \u003cem\u003eAcinetobacter\u003c/em\u003e species identification has been well established for over a decade. For instance, Lee et al. (2014) demonstrated its superior accuracy compared to automated methods such as Vitek2 (90.5% vs. 100% concordance with Kraken 2 and ANI). Thus, gyrB targeted PCR remains a reliable and essential tool for epidemiological studies involving clinical \u003cem\u003eAcinetobacter\u003c/em\u003e strains [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOf the isolates analyzed, 71.42% (30/42) were confirmed as \u003cem\u003eA. baumannii\u003c/em\u003e (Table\u0026nbsp;1). Differentiating species within the ACB complex is critical but challenging due to their close genetic relationships and phenotypic similarities [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. It should be noted that the blaOXA-51-like gene can also be used to identify \u003cem\u003eA. baumannii\u003c/em\u003e, as it has been found in virtually every isolate of this species within the ACB complex [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. In fact, combined identification methods are often required; therefore, PCR amplification of the gyrB gene and MALDI-TOF mass spectrometry represent optimal approaches for identifying members of the \u003cem\u003eAcinetobacter calcoaceticus-A. baumannii\u003c/em\u003e complex [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eClonal Diversity Analysis\u003c/p\u003e \u003cp\u003eThe spread of resistant bacterial clones in healthcare settings poses a significant threat to public health by complicating treatment and increasing patient morbidity and mortality. Accurate clone identification is critical for infection control, outbreak tracking, and the development of prevention strategies. Molecular biology tools such as PCR facilitate the mapping of genetic diversity and resistance profiles, aiding personalized treatment and health policy development [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOf the 42 \u003cem\u003eAcinetobacter\u003c/em\u003e spp. isolates analyzed, 23 (55%) \u003cem\u003eA. baumannii\u003c/em\u003e strains were selected as a representative subset for clonal profiling, ensuring coverage of variation in both clinical origin and colistin resistance and susceptibility. This subset included 3 isolates from tracheal secretions, 4 from tracheal aspirates, 3 from urine, 5 from catheter tip samples, 5 from blood cultures, and 3 from skin secretions. Of these, 14 were colistin-resistant and 9 were colistin-sensitive. The selection of these isolates was not based solely on colistin resistance but rather on their identification as \u003cem\u003eA. baumannii\u003c/em\u003e and the need to represent the diversity of clinical sources and epidemiological characteristics within the collection.\u003c/p\u003e \u003cp\u003eTwo \u003cem\u003eA. baumannii\u003c/em\u003e clones were identified in clusters 1 (12 strains) and 2 (7 strains), with isolates exhibiting both colistin-sensitive and colistin-resistant profiles (Fig.\u0026nbsp;2). Cluster 3 (4 strains) consisted of species belonging to the ACB complex. The dominant clones were associated with HC-UFMG (Group 1) and Hospital Jo\u0026atilde;o XXIII, both located in Belo Horizonte, Minas Gerais (Group 2).\u003c/p\u003e \u003cp\u003eClone identification is crucial for understanding hospital infection dynamics and selecting appropriate antimicrobial therapies. The limited number of clones identified may reflect a recent population contraction of \u003cem\u003eA. baumannii\u003c/em\u003e, possibly due to its narrow ecological niche. Unlike other \u003cem\u003eAcinetobacter\u003c/em\u003e species with broad environmental distribution, \u003cem\u003eA. baumannii\u003c/em\u003e is primarily associated with human infections, which may restrict its genetic diversity [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOver the past three decades, \u003cem\u003eA. baumannii\u003c/em\u003e has gained clinical relevance due to its capacity to acquire and express antimicrobial resistance genes. Strains belonging to the ACB complex (Groups 1 and 2) in cluster 3 included both sensitive and colistin-resistant isolates. The observed clonal diversity can be attributed to antimicrobial pressure, adaptation to hospital environments, and efficient transmission among patients and surfaces. This pathogen is frequently associated with nosocomial infections, particularly in intensive care units (ICUs), where antibiotic misuse and failures in infection control enhance its persistence [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eClone 1 isolates were predominantly obtained from tracheal secretions (bacterial pneumonia) and catheter tips (bloodstream infections), whereas clone 2 isolates were mainly recovered from tracheal aspirates (bacterial pneumonia) and blood cultures (bloodstream infections) (Fig.\u0026nbsp;2). Most \u003cem\u003eA. baumannii\u003c/em\u003e bloodstream infections are associated with medical care [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], while bacterial pneumonia caused by \u003cem\u003eA. baumannii\u003c/em\u003e is linked to high mortality and morbidity, especially in critically ill ICU patients [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The data from this study indicate that both clones 1 and 2 are involved in these infections, suggesting that they may share similar virulence factors that enable colonization and infection of these sites.\u003c/p\u003e \u003cp\u003eWhen considering colistin resistance, clone 2 comprised isolates with higher resistance rates (6 R / 1 S) compared to clone 1 (5 R / 7 S) (p\u0026thinsp;=\u0026thinsp;0.05) (Fig.\u0026nbsp;2). The detection of clone 2, characterized by a higher proportion of resistant isolates, underscores the importance of monitoring infections in hospital settings. Previous studies have identified the ST2 global clone as the predominant CRAB lineage in ICUs [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eColistin Minimum Inhibitory Concentration (MIC)\u003c/p\u003e \u003cp\u003eColistin susceptibility was assessed via broth microdilution following BrCAST (2024) criteria (Table\u0026nbsp;1). \u003cem\u003eA. baumannii\u003c/em\u003e is commonly associated with severe infections in immunosuppressed ICU patients [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this study, isolates from tracheal aspirates, catheter tips, and blood cultures showed high levels of colistin resistance (26 resistant strains; MIC\u0026thinsp;\u0026gt;\u0026thinsp;4 \u0026micro;g/mL).\u003c/p\u003e \u003cp\u003eAlthough polymyxins (e.g., colistin) serve as last-line treatments, their overuse has contributed to the emergence of resistance, posing a major public health concern [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, 61.90% (26/42) of isolates were resistant to colistin, with Group 1 showing 75% resistance compared to 38.9% in Group 2. MIC values ranged from 4 to 32 \u0026micro;g/mL. Approximately 70% of isolates from bacterial pneumonia (7/10) and 64.0% of bloodstream infection isolates (9/14) were resistant to colistin (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eAmong colistin-resistant \u003cem\u003eA. baumannii\u003c/em\u003e isolates, Group 1 had a higher number of resistant strains (15/18) than Group 2 (3/12) (p\u0026thinsp;=\u0026thinsp;0.0024). Colistin-resistant \u003cem\u003eA. baumannii\u003c/em\u003e strains are often associated with pneumonia and bloodstream infections and represent a major cause of nosocomial infections with significant morbidity and mortality. This pattern is consistent with previous reports of CRAB infections, in which mortality rates particularly among patients with pneumonia and bloodstream infections may reach approximately 60% [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiofilm Cell Density Quantification\u003c/p\u003e \u003cp\u003eIn this study, significant differences in biofilm cell density were observed between colistin-resistant and colistin-sensitive \u003cem\u003eA. baumannii\u003c/em\u003e strains. Resistant strains formed denser biofilms (5.02 \u0026times; 10\u0026sup1;\u0026sup1; CFU/mL) than sensitive strains (4.9 \u0026times; 10\u0026sup1;⁰ CFU/mL; p\u0026thinsp;=\u0026thinsp;0.0173) (Graph 1A). This difference may be related to mechanisms such as antimicrobial defense [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], metabolic adaptation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], increased structural protection [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and interspecies interactions [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe combination of antimicrobial resistance and biofilm formation makes \u003cem\u003eA. baumannii\u003c/em\u003e a formidable pathogen in healthcare settings, where it can cause pneumonia, bloodstream infections, wound infections, and urinary tract infections [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Biofilms provide a protective environment for bacterial cells and further enhance their resistance to antimicrobial agents; they are also associated with persistent infections and the development of chronic disease.\u003c/p\u003e \u003cp\u003eWithin the ACB complex, no significant differences in biofilm density were observed between colistin-resistant (1.92 \u0026times; 10\u0026sup1;⁰ CFU/mL) and sensitive strains (1.25 \u0026times; 10\u0026sup1;\u0026sup1; CFU/mL; p\u0026thinsp;\u0026ge;\u0026thinsp;0.1). Factors such as surface adhesion [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], nutrient availability [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], humidity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], physicochemical conditions, and quorum sensing [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] influence biofilm development.\u003c/p\u003e \u003cp\u003ePrevious studies (Kaplan, 2011; Sato et al., 2018; Wang et al., 2010) have shown that subinhibitory antibiotic concentrations can stimulate biofilm formation and increase bacterial virulence. Consistent with these findings, sensitive \u003cem\u003eA. baumannii\u003c/em\u003e strains exposed to subinhibitory colistin concentrations produced significantly denser biofilms (4.6 \u0026times; 10\u0026sup1;\u0026sup1; CFU/mL) than unexposed strains (4.9 \u0026times; 10\u0026sup1;⁰ CFU/mL; p\u0026thinsp;=\u0026thinsp;0.0136) (Graph 1B).\u003c/p\u003e \u003cp\u003eAmong the identified clones, clone 2 exhibited the highest biofilm density under antimicrobial pressure, followed by clones 1 and 3 (Graph 1C). The enhanced biofilm-forming capacity of clone 2 under colistin pressure (1.29 \u0026times; 10\u0026sup1;⁴ CFU/mL) suggests increased virulence and persistence, representing a significant public health concern.\u003c/p\u003e \u003cp\u003eIsolates with higher biofilm production were mainly obtained from tracheal aspirates and tracheal secretions (bacterial pneumonia) (Graph 1D). \u003cem\u003eA. baumannii\u003c/em\u003e exhibits increased biofilm formation at the solid liquid interface, as observed in pneumonia cases. Previous studies have shown that isolates from patients with aspiration pneumonia produce substantial amounts of biofilm [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDetection of the bap Gene\u003c/p\u003e \u003cp\u003eThe bap gene, an important virulence factor in \u003cem\u003eA. baumannii\u003c/em\u003e, was screened in all isolates to evaluate its association with biofilm formation (Fig.\u0026nbsp;4). All \u003cem\u003eA. baumannii\u003c/em\u003e and ACB complex isolates carried the bap gene, indicating a 100% prevalence among hospital isolates. In another study, the prevalence of bap was 79.2%, and a significant correlation was observed between antibiotic resistance, biofilm formation, and biofilm-associated genes [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe detection of bap in all clinical specimens analyzed suggests that this gene contributes to biofilm formation, persistence in hospital environments and on medical devices, and the pathogenesis of healthcare associated infections. Its presence was confirmed in both antibiotic-resistant and antibiotic-sensitive isolates.\u003c/p\u003e \u003cp\u003eThis gene promotes adhesion and colonization in hospital environments, thereby enhancing persistence and resistance to disinfectants and host immune responses [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Understanding the role of bap is crucial for developing new therapeutic strategies, as previous studies have demonstrated that bap expression leads to robust biofilm formation and increased resistance to antimicrobial treatments [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to De Gregorio et al. (2015), Bap is highly polymorphic, and this variability may influence biofilm formation in Gram-negative bacteria [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Further molecular studies investigating Bap and its interaction with other proteins may help clarify the differences in biofilm-forming capacity observed between \u003cem\u003eA. baumannii\u003c/em\u003e and ACB complex isolates.\u003c/p\u003e \u003cp\u003eSCV Formation Under Colistin Pressure\u003c/p\u003e \u003cp\u003eSmall colony variants (SCVs) are slow-growing bacterial subpopulations characterized by altered metabolism and increased persistence [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Their formation was evaluated in both \u003cem\u003eA. baumannii\u003c/em\u003e and ACB complex isolates, with and without exposure to subinhibitory concentrations of colistin (Graph 1E and 1F).\u003c/p\u003e \u003cp\u003eColistin-resistant \u003cem\u003eA. baumannii\u003c/em\u003e strains produced more SCVs without antibiotic pressure (5.4 \u0026times; 10⁻\u0026sup2; CFU/mL) than with subinhibitory colistin (1.2 \u0026times; 10⁻\u0026sup2; CFU/mL; p\u0026thinsp;=\u0026thinsp;0.025). Conversely, colistin-sensitive strains produced more SCVs under subinhibitory colistin exposure (5 \u0026times; 10⁻\u0026sup2; CFU/mL) than without exposure (1 \u0026times; 10⁻\u0026sup2; CFU/mL; p\u0026thinsp;=\u0026thinsp;0.035).\u003c/p\u003e \u003cp\u003eThese results suggest that subinhibitory concentrations of colistin can promote biofilm formation in sensitive strains and stimulate the emergence of SCVs, highlighting the potential risks associated with subtherapeutic antimicrobial exposure. These findings are consistent with previous studies showing that subinhibitory antibiotic concentrations can induce the SOS response and increase SCV formation [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe frequency of SCV generation was significantly higher in colistin-resistant \u003cem\u003eA. baumannii\u003c/em\u003e strains (5.4 \u0026times; 10⁻\u0026sup2;) compared to susceptible strains (1 \u0026times; 10⁻\u0026sup2;) (p\u0026thinsp;=\u0026thinsp;0.0226). Previous studies have shown that phase variation in \u003cem\u003eA. baumannii\u003c/em\u003e contributes to resistance against several antibiotics, including variations in colistin MIC values [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSCVs are particularly concerning because they require 48\u0026ndash;72 hours to grow and are often undetectable by conventional microbiological methods, representing a significant public health concern. Their increased antibiotic resistance allows them to persist in environments with high antimicrobial pressure, such as hospitals, contributing to infections that are difficult to treat [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSCV Species Confirmation by gyrB PCR\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e SCVs exhibited altered band patterns compared to their parental strains (Fig.\u0026nbsp;5). Previous studies have shown that colony phase variation in \u003cem\u003eA. baumannii\u003c/em\u003e the process that generates opaque (virulent) and translucent (avirulent) colonies is regulated by DNA rearrangements involving recombinase machinery and DNA methylation-associated epigenetic modifications [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe formation of SCVs in \u003cem\u003eA. baumannii\u003c/em\u003e may involve alterations in the gyrB gene sequence as well as DNA rearrangements mediated by recombinase activity and epigenetic modifications linked to DNA methylation. The gyrB gene, together with gyrA, encodes DNA gyrase, which plays a crucial role in DNA replication, transcription, and recombination [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Therefore, the altered band patterns observed in the gyrB gene of SCVs are likely due to sequence variations and should be further investigated to clarify the molecular mechanisms underlying SCV formation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated that colistin-resistant strains of \u003cem\u003eA. baumannii\u003c/em\u003e produce significantly higher biofilm cell densities and exhibit increased formation of SCVs, both of which are associated with enhanced resistance and persistence in hostile environments.\u003c/p\u003e \u003cp\u003eIn conclusion, the ability of \u003cem\u003eA. baumannii\u003c/em\u003e to form dense biofilms, combined with SCVs formation and clonal diversity, represents a significant challenge in the treatment of HAIs. The global dissemination of high risk clones underscores the urgent need for effective prevention and control strategies. These findings reinforce the need for robust infection control measures, antimicrobial stewardship, and the development of targeted therapies to mitigate the threat posed by colistin resistant \u003cem\u003eA. baumannii\u003c/em\u003e in healthcare environments. Furthermore, intensified research into resistance mechanisms, novel therapeutic approaches, and strict surveillance measures in healthcare settings is essential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding Information\u003c/h2\u003e \u003cp\u003eUFMG Support Foundation (FUNDEP) Grant Number: \u003cb\u003e30201*52.\u003c/b\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLarissa Naneti Rosa, Magna Cristina de Paiva, and Santiago Lattar drafted the main manuscript text. Giovanna Costa da Silva and Sophia Mendes prepared Figures 1\u0026ndash;5 and Graph 1. All authors contributed to the revision of the manuscript and approved the final version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIbrahim S, Al-Saryi N, Al-Kadmy IMS, Aziz SN (2021) Multidrug-resistant Acinetobacter baumannii as an emerging concern in hospitals. Mol Biol Rep 48(10):6987\u0026ndash;6998. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11033-021-06690-6\u003c/span\u003e\u003cspan address=\"10.1007/s11033-021-06690-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Wang Y, Zhang N, Li J, Liu X (2024) Genotypic and phenotypic characteristics of Acinetobacter baumannii isolates from the people's hospital of Qingyang City, Gansu province. 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PLoS Pathog 11(4):e1004870. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.ppat.1004870\u003c/span\u003e\u003cspan address=\"10.1371/journal.ppat.1004870\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n"},{"header":"Graph 1","content":"\u003cp\u003eGraph 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acinetobacter baumannii, SCVs, biofilms, clonal diversity","lastPublishedDoi":"10.21203/rs.3.rs-9094954/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9094954/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e is a Gram-negative bacterium frequently associated with healthcare-associated infections (HAIs) and represents a growing public health concern due to its resistance to multiple antimicrobials. \u003cem\u003eA. baumannii\u003c/em\u003e is particularly notable for its ability to form biofilms, complex cellular structures adhered to surfaces, and for its clonal diversity, which complicates infection control. Additionally, the formation of small colony variants (SCVs) has been associated with increased virulence and resistance. This study aimed to analyze biofilm formation and SCV occurrence among \u003cem\u003eA. baumannii\u003c/em\u003e clones, as well as to investigate the presence of the bap gene, which is associated with biofilm production. Biofilms were developed under static conditions in 6-well plates at 37\u0026deg;C for 24 hours. Results showed that colistin-resistant \u003cem\u003eA. baumannii\u003c/em\u003e strains produced significantly higher biofilm cell densities (5.02 \u0026times; 10\u0026sup1;\u0026sup1; CFU/mL) compared to susceptible strains (4.9 \u0026times; 10\u0026sup1;⁰ CFU/mL; p\u0026thinsp;=\u0026thinsp;0.0173). Moreover, resistant strains exhibited a higher frequency of SCV generation (5.4 \u0026times; 10⁻\u0026sup2;) than susceptible isolates (1 \u0026times; 10⁻\u0026sup2;; p\u0026thinsp;=\u0026thinsp;0.0226). Three distinct \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e and \u003cem\u003eAcinetobacter calcoaceticus\u0026ndash;A. baumannii\u003c/em\u003e (ACB) complex clones were identified using Random Amplified Polymorphic DNA (RAPD) analysis. Clone 2 produced the highest biofilm cell density under subinhibitory colistin concentration (1.29 \u0026times; 10\u0026sup1;⁴ CFU/mL), which was significantly greater than that of Clone 1 (5.22 \u0026times; 10\u0026sup1;\u0026sup1; CFU/mL; p\u0026thinsp;=\u0026thinsp;0.033) and Clone 3 (1.79 \u0026times; 10\u0026sup1;\u0026sup1; CFU/mL; p\u0026thinsp;=\u0026thinsp;0.021). Additionally, the bap gene was detected in all isolates, suggesting its important role in adhesion and persistence. In conclusion, the ability of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e to form dense biofilms, together with the emergence of small colony variants (SCVs), represents a major challenge for the effective treatment and control of healthcare-associated infections (HAIs). These adaptive traits contribute to increased persistence in clinical environments, enhanced tolerance to antimicrobial agents, and difficulties in eradication, highlighting the need for improved strategies to prevent and manage these infections.\u003c/p\u003e","manuscriptTitle":"Analysis of Biofilm Formation and Small Colony Variant Development under Colistin Pressure in Different Clones of Acinetobacter spp","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 19:00:29","doi":"10.21203/rs.3.rs-9094954/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"427a982a-6ea4-4c63-a0be-1d19cf41bf42","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-29T12:39:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 19:00:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9094954","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9094954","identity":"rs-9094954","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}