High Biofilm-forming Capacity Research Articles

Role of <i>Actinomyces</i> species in oral Biofilm Formation and Dental Plaque-Related Diseases

Introduction: Actinomyces species play a major role in the development of oral biofilms and disorders associated with dental plaque. The purpose of this study was to look into their interactions, prevalence, and ability to form biofilms in dental plaque. Methodology: Dental plaque samples from 78 participants in a six-month cross-sectional research at KCD Peshawar were examined for Actinomyces species. The microtiter plate test was used to measure the production of biofilms, and statistical analyses were performed to investigate the relationships between Actinomyces presence, plaque severity, and biofilm formation capacity. Results: A total of 79.5% of samples contained Actinomyces species, with Actinomyces naeslundii being the most common (72.6%). Actinomyces was found to be significantly correlated with the severity of the plaque (χ² = 8.92, p = 0.012). In comparison to mild/moderate plaque (mean OD570 = 0.52, t(60) = 2.78, p = 0.007), isolates from severe plaque (mean OD570 = 0.61) formed more biofilms. Increased biofilm biomass was obtained through co-cultivation with Streptococcus mutans, particularly in combinations involving A. israelii and S. mutans. Conclusion: Actinomyces species are important in the development and severity of dental plaque, with more severe cases being associated with higher biofilm-forming capacity. Their relationships to Streptococcus mutans increase the pathogenicity of biofilms. Actinomyces targeting may be able to reduce tooth plaque-related illnesses. Larger populations and biological reasons for successful interventions should be investigated in future studies.

Listeria monocytogenes from Food Products and Food Associated Environments: Antimicrobial Resistance, Genetic Clustering and Biofilm Insights.

Listeria monocytogenes, a foodborne pathogen, exhibits high adaptability to adverse environmental conditions and is common in the food industry, especially in ready-to-eat foods. L. monocytogenes strains pose food safety challenges due to their ability to form biofilms, increased resistance to disinfectants, and long-term persistence in the environment. The aim of this study was to evaluate the presence and genetic diversity of L. monocytogenes in food and related environmental products collected from 2014 to 2022 and assess antibiotic susceptibility and biofilm formation abilities. L. monocytogenes was identified in 13 out of the 227 (6%) of samples, 7 from food products (meat preparation, cheeses, and raw milk) and 6 from food-processing environments (slaughterhouse-floor and catering establishments). All isolates exhibited high biofilm-forming capacity and antibiotic susceptibility testing showed resistance to several classes of antibiotics, especially trimethoprim-sulfamethoxazole and erythromycin. Genotyping and core-genome clustering identified eight sequence types and a cluster of three very closely related ST3 isolates (all from food), suggesting a common contamination source. Whole-genome sequencing (WGS) analysis revealed resistance genes conferring resistance to fosfomycin (fosX), lincosamides (lin), fluoroquinolones (norB), and tetracycline (tetM). In addition, the qacJ gene was also detected, conferring resistance to disinfecting agents and antiseptics. Virulence gene profiling revealed the presence of 92 associated genes associated with pathogenicity, adherence, and persistence. These findings underscore the presence of L. monocytogenes strains in food products and food-associated environments, demonstrating a high virulence of these strains associated with resistance genes to antibiotics, but also to disinfectants and antiseptics. Moreover, they emphasize the need for continuous surveillance, effective risk assessment, and rigorous control measures to minimize the public health risks associated to severe infections, particularly listeriosis outbreaks. A better understanding of the complex dynamics of pathogens in food products and their associated environments can help improve overall food safety and develop more effective strategies to prevent severe health consequences and economic losses.

A prospective study of biofilm production among catheterized and non-catheterized urinary bacterial isolates along with their antimicrobial susceptibility pattern in tertiary care hospital, Nadiad, Gujarat

Background: Urinary tract infections (UTIs) are one of the leading causes of morbidity, mortality, and health-care expenditure, affecting all age groups of people. Bacteria that are able to produce biofilm are more likely to cause recurrent UTI. Conversely, bacteria with higher biofilm-forming capacity are much more likely to cause recurrent UTI. Understanding biofilm formation and antimicrobial resistance determinants of uropathogenic organisms will provide insight into the development of treatment options for biofilm-associated UTIs. Aim and Objectives: The objectives of this study are to compare antimicrobial resistance patterns among biofilm-producing and non-biofilm-producing organisms isolated from catheterized and non-catheterized patients with UTIs. Hence, we can improve patient outcomes, reduce antimicrobial resistance, and decrease the spread of infections caused by multidrug-resistant organisms of biofilm-associated UTIs in all age groups of catheterized and non-catheterized patients visiting Dr. N. D. Desai Faculty of Medical Science and Research, Nadiad, Gujarat, during the study period. Materials and Methods: A prospective study was done to check the prevalence of biofilm production in catheterized and non-catheterized patients and their antimicrobial susceptibility pattern isolated from urine samples for a period of 1 year (from July 2022 to June 2023) at the Department of Microbiology, Dr. N. D. Desai Faculty of Medical Science and Research, Nadiad, Gujarat. The results of the microbiological profile and their susceptibility pattern were tabulated and statistically analyzed. Results: Out of 1360 urine samples, 370 samples grew pathogen. (GPC: 67, GNB: 319, and 16 of them grew 2 pathogens). Biofilm production was found in 78 (20.2%) of pathogens isolated from catheterized and non-catheterized patients. Commonly isolated pathogen was Gram-negative bacilli 82.6% followed by Gram-positive cocci 17.4%. Escherichia coli (67.4%) was the most common isolate found followed by Enterococcus spp. (13.5%), Klebsiella spp. (6.2%), Pseudomonas spp. (4.2%), Staphylococcus spp. (2.7%), Proteus spp. (2%), Enterobacter spp. (1.3%), Acinetobacter spp. (1.3%), Staphylococcus aureus (1.3%), and Citrobacter spp. (0.2%). Conclusion: UTIs are the most common infection in the community as well as health-care-associated infections. A biofilm-producing pathogen is more resistant to antimicrobial agents. Hence, it is necessary to detect biofilm formation at an early stage along with an antimicrobial susceptibility test to decrease the usage of last resort antimicrobial agents and the high costs of individual’s health as well as the health-care system around the world, particularly in developing countries.

Phenotypical Comparison between Environmental and Clinical Acinetobacter baumannii Strains Isolated from an Intensive Care Unit.

Acinetobacter baumannii (A. baumannii) causes a variety of nosocomial infections that mainly affect critically ill patients in intensive care units (ICUs). The objective of this study was to assess the prevalence of A. baumannii in the ICU environment and evaluate the antibiotic resistance and biofilm formation ability of the environmental isolates compared to those isolated from ICU patients simultaneously. A total of 166 non-duplicate ICU samples (80 environmental and 86 clinical) were collected between January 2019 and January 2020. Antimicrobial susceptibility detection was determined using the disc diffusion method, and the strains were evaluated for the minimum inhibitory concentration (MIC) of imipenem (IMP) using broth microdilution or metallo-β-lactamase (MBL) detection according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The isolates' capacity to produce biofilms was evaluated using the tube method and the crystal violet microtitre plate-based method. A. baumannii was identified in 25 (31.25%) environmental and 30 (34.88%) clinical samples, and beds were the most infected (60%). Both types of isolate demonstrated a rate surpassing 80% resistance to the tested antibiotics. Phenotypically, the environmental and clinical strains were found to be MBL producers. Fourteen environmental (56%) and 15 clinical (50%) strains were found to be moderate biofilm producers, indicating that each isolate has a high biofilm-forming capacity. These results show that the spread of multidrug-resistant (MDR) A. baumannii in an ICU setting emphasises the necessity of disinfecting and cleaning medical devices and surfaces to prevent and restrict cross-transmission. Intensive surveillance and infection control methods are also of paramount importance.

Evaluation of Malassezia furfur Biofilm Formation on Polypropylene Membrane

The Malassezia yeast species colonize on the skin immediately after birth and could be found on the healthy skin flora for life. Although they are more frequently involved in the etiology of common skin infections in the community, particularly Malassezia furfur could cause life-threatening infections such as fungemia. Detection of biofilm during the colonization of these yeasts on the skin is an important criterion for its virulence. Since they are lipophilic yeasts, commonly used biofilm detection methods are not applicable to the Malassezia strains. The aim of the study was to describe the growth and measurement of M.furfur isolates on a polypropylene membrane to demonstrate their biofilm-forming capacities. Twenty-seven M.furfur strains colonized in the newborns were included in the study. Basically, sterile polypropylene membranes were placed on different polysorbates (tween 20, 40, and 80) which were spread on Sabouraud dextrose agar. Ten µl saline suspension of M.furfur was dropped on the polypropylene membrane and incubated in standard growth conditions for three days. Later, the visible colony was removed gently by washing with running water and the biofilm structure formed on the membrane was stained with safranin. The stained biofilm was photographed. Performing image analysis, the values obtained against background activity were digitized according to the specified protocol. Moreover, XTT reduction test was performed and the measured metabolic activity results were compared with the safranin-stained biofilm data. The safranin hydrolysis of the strains was measured spectrometrically. Twenty-five (92.6%) of the strains included in the study were stained with safranin, which indicated the presence of biofilm on the polypropylene membrane. The strains grown with tween 20 and tween 80 formed a higher biofilm layer density than those supplied with tween 40. Isolates with low and high biofilm-forming capacity were clearly separated by tween 20 (p< 0.05). XTT activity was detected in 26 (96.3%) isolates. No correlation was found between biofilm density obtained by the described method and XTT reduction. It was observed that hydrolysis of safranin did not affect the biofilm evaluation method. In this study, it was shown that as a result of sufficient diffusion through hydrophobic membranes, polysorbate-based growth factors could maintain measurement of the biofilm layer formed by lipophilic M.furfur strains. The best grouping properties for M.furfur were obtained with tween 20 which could determine low and high level of biofilm formation. Image analysis was used with high performance for this method. As conclusion, the utilization of different hydrophobic membranes and dyes would lead to the development of new techniques for the application in other lipophilic yeasts.

Bacteriological Profile of Chronic Rhinosinusitis and Adenotonsillitis: Evaluating the Role of Biofilm Production and Multidrug Resistance.

There is significant evidence that the etiology of chronic otorhinolaryngology infections such as chronic rhinosinusitis, adenotonsillitis, and otitis media depends on biofilms. As biofilm-forming bacteria can be resistant to the immune system, antibiotics, and other treatments, biofilm infections are often chronic. To identify the genus and species of the clinical isolates obtained from the swabs collected from the patients with chronic infections of the nasal and paranasal sinus, nasopharynx, and oropharynx and to evaluate phenotypic and genotypic methods for the detection of biofilms and antimicrobial resistance among the isolated organisms. A total of 100 patients with chronic rhinosinusitis and adenotonsillitis participated in this study. Various clinical samples from the nasal cavity, nasopharynx, and oropharynx were obtained and subjected to microbiological analysis and biofilm-forming capacity by three methods: tube methods, Congo red staining, and microtiter plate method. The various specific genes were amplified by polymerase chain reaction. The amplified gene products were separated by gel electrophoresis. This was a prospective cohort study conducted on a total of 100 patients with chronic rhinosinusitis and adenotonsillitis. The age of the study participants was between 7 and 53years with a mean age of 29.22 ± 15.03. This study included 54 (54%) nasal tissue samples and 46 (46%) adenotonsillar tissue. The frequently cultured organisms are coagulase-negative staphylococci (17%), E. coli (10%), Citrobacter (10%), and Klebsiella (7%). Staphylococcus aureus (4), and Methicillin-resistant Staphylococcus aureus (3) produced strong biofilm. Acenobacter (3), Citrobacter (4), and E. coli (4) showed moderate biofilm production. Coagulase-negative Staphylococcus aureus (11), E. coli (6), and Klebsiella (7) showed weak biofilm formation. Citrobacter (6), and Coagulase negative Staphylococcus aureus (6) were negative for biofilm production. Staphylococcus aureus expressed mecA gene (3) and Panton-Valentine Leukocidin gene (2), Pseudomonas expressed mucA gene (2), Citrobacter expressed blaCARB-2 (4) qnrA gene (2), E. coli expressed bla SHV (2) and bla TEM1 gene (2) and Klebsiella expressed Kfu (2) and uge (1). Acenobacter was negative for blaIMP1, blaVIM2 genes. This study adds to the information on the common pathogens-forming biofilms in various nasal pathologies and adenotonsillitis. The knowledge that a particular organism has a higher biofilm-forming capacity will help to sensitize the physician that factors such as biofilms may be at play and take appropriate measures.

Biofilm formation, antibiotic resistance, and genome sequencing of a unique isolate Salmonella Typhimurium M3

Salmonella Typhimurium is a zoonotic bacterium that can cause salmonellosis, and the major concerns of S. Typhimurium for the food industry are its ability to obtain multidrug resistance and form biofilms on food-contact surfaces. In the current study, the antimicrobial resistance of a strong biofilm former S. Typhimurium M3 was assessed by the diffusion method. Genome sequencing was also applied to obtain the genes related to antibiotic resistance, and biofilm formation of S. Typhimurium M3. Biofilm-forming capacity of S. Typhimurium M3 was found to be strain dependent, and a high number of isolates were strong biofilm formers. The high biofilm-forming isolate S. Typhimurium M3 was resistant to oxacillin, lincomycin, rifampicin, tetracycline, and clindamycin, with the MIC values of 512 μg/mL, 32 μg/mL, 16 μg/mL, 64 μg/mL, and 64 μg/mL, respectively. Genomic annotation of S. Typhimurium M3 showed the presence of genes involved in cellulose biosynthesis, curli production, fimbriae biosynthesis, flagellar assemble, quorum sensing, chemotaxis, and some transcriptional regulators. Antibiotic efflux conferring antibiotic resistance genes, antibiotic inactivation genes, and antibiotic target alteration genes were also identified. The results expand scientific understanding on how Salmonella isolates with high biofilm-forming capacity and multidrug resistance survive in stressful conditions in the industry.

Cell Immunity in Implant-Associated Infections Caused by Biofilm-Forming Microorganisms.

High biofilm-forming capacity of Staphylococcus spp. strains isolated from biomaterial of patients with infectious complications after primary knee replacement developed within 6-12 months after surgery was experimentally demonstrated. Differential leukocyte counts and some indicators of cell immunity in these patients were compared with those in patients without purulent complications and healthy volunteers. In patients with implant-associated infection, the relative numbers of T cells (both T-helpers and T-suppressors) B cells were significantly (p<0.05) reduced, while the number of NK cells was significantly increased in comparison with the corresponding parameters in other groups. The revealed changes attest to cell immunity failure in biofilm infection.

Draft Genome Sequence of the Urinary Catheter Isolate Enterobacter ludwigii CEB04 with High Biofilm Forming Capacity.

Enterobacter ludwigii is a fermentative Gram-negative environmental species and accidental human pathogen that belongs to the Enterobacter cloacae complex with the general characteristics of the genus Enterobacter. The clinical isolate E. ludwigii CEB04 was derived from a urinary tract catheter of an individual not suffering from catheter-associated urinary tract infection. The draft genome sequence of the high biofilm forming E. ludwigii CEB04 was determined by PacBio sequencing. The chromosome of E. ludwigii CEB04 is comprised of one contig of 4,892,375 bps containing 4596 predicted protein-coding genes and 120 noncoding RNAs. E. ludwigii CEB04 harbors several antimicrobial resistance markers and has an extended cyclic-di-GMP signaling network compared to Escherichia coli K-12.

Whole genome sequence of blaNDM and blaKPC co-producing Klebsiella pneumoniae isolate KSH203 with capsular serotype K25 belonging to ST11 from China.

The aim of this study was to characterise a high biofilm-forming capacity, hypermucoviscous, blaKPC and blaNDM co-producing Klebsiella pneumoniae strain (KSH203). Antimicrobial susceptibility, biofilm formation and hypermucoviscous phenotype were determined by the disk diffusion method, crystal violet staining and positive string test, respectively. Whole-genome sequencing was performed using a PacBio RS II Sequencer. High-quality reads were de novo assembled using Celera Assembler v.8.0. Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and the genome characteristics were analysed by bioinformatics methods. Klebsiella pneumoniae strain KSH203 was resistant to all antibiotics tested but was only intermediate-resistant to polymyxin B. This strain showed high biofilm-forming ability and a hypermucoviscous phenotype with serotype K25 belonging to the ST11 clone. KSH203 consists of a 5 464 059-bp single chromosome and four plasmids including pKSH203-NDM (53 144 bp), pKSH203-KPC (159 467 bp), pKSH203-CTX-M-3 (156 910 bp) and pKSH203-qnrS (253 705 bp). A total of 44 antimicrobial resistance genes and a large number virulence-associated genes were identified in the genome of strain KSH203. In this study, we illustrate the whole genome sequence of high biofilm-forming capacity, hypermucoviscous K. pneumoniae isolate KSH203 with capsular serotype K25 belonging to ST11 isolated from a patient in China, which carried a large number of antimicrobial resistance genes and virulence-associated genes. Future studies are needed to be aware of dissemination of this type of strain among environmental, animal and human isolates.

Candida parapsilosis Sensu Stricto: A Pathobiont in Conditions of Oral Dysbiosis?

Recent publications have reported high prevalence of C. parapsilosis sensu stricto in oral cavity niches. Our research group performed a pilot study in 2017 which showed that of the psilosis complex, C. parapsilosis sensu stricto is the species most frequently isolated from oral cavity niches. Under inflammatory conditions, the probability of recovering it is almost four times higher, and it displays higher biofilm-forming capacity in-vitro, differing significantly from isolates of the same species obtained in conditions of eubiosis. This led us to hypothesize that an oral environment in dysbiosis overregulates virulent genes, promoting a more pathogenic phenotype. Accordingly, we decided to reevaluate the in-vitro biofilm formation assay using a larger sample, under two nutritional conditions, using colorimetric methods for quantification. In addition, the results obtained were validated by imaging techniques. For both clinical conditions (eubiosis and dysbiosis), high biofilm forming phenotype predominated with both reading methods, and for both nutritional conditions tested. XTT showed significant difference between absorbance values for isolates from buccal dysbiosis and eubiosis (p=0.0025). C. parapsilosis sensu stricto cells that colonize oral cavity niches are basically strong biofilm formers, regardless of in vitro growing conditions. However, the oral cavity in dysbiosis probably promotes virulence in this species due to inheritable epigenetic modifications.

Frequent carriage of resistance mechanisms to β-lactams and biofilm formation in Haemophilus influenzae causing treatment failure and recurrent otitis media in young children.

Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry resistance mechanisms to β-lactams. A collection of 48 H. influenzae isolates was obtained by tympanocentesis or from otorrhoea samples from individual patients <3 years of age and diagnosed with recurrent or treatment failure AOM. Each isolate was surveyed for the presence of blaTEM genes, amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3) and biofilm formation in microtitre plates. In 43 of the 48 isolates (89.6%), at least one of the three tested conditions was identified: biofilm formation (83.3%) and resistance mechanisms to β-lactams (33.3%), modifications in the transpeptidase domain of PBP3 being the most prevalent (22.9%), followed by β-lactamase production (10.4%). Additionally, 13 (27.1%) isolates had two or more of these three traits. In relation to biofilm formation, those isolates with an amoxicillin MIC ≤ 0.5 mg/L had higher optical density values than isolates with an amoxicillin MIC ≥ 1 mg/L (Mann-Whitney U-test, P=0.048). These findings suggest that the successful treatment of non-typeable H. influenzae causing chronic and recurrent AOM in young children may be compromised by the high biofilm-forming capacity of the isolates and the presence of β-lactam resistance mechanisms, particularly PBP3 mutations.

Is biofilm formation related to the hypermutator phenotype in clinicalEnterobacteriaceaeisolates?

In bacteria, complex adaptive processes are involved during transition from the planktonic to the biofilm mode of growth, and mutator strains are more prone to producing biofilms. Enterobacteriaceae species were isolated from urinary tract infections (UTIs; 222 strains) and from bloodstream infections (BSIs; 213 strains). Relationship between the hypermutable phenotype and biofilm forming capacity was investigated in these clinical strains. Mutation frequencies were estimated by monitoring the capacity of each strain to generate mutations that conferred rifampicin resistance on supplemented medium. Initiation of biofilm formation was assayed by determining the ability of the cells to adhere to a 96-well polystyrene microtitre plate. UTI Enterobacteriaceae strains showed significantly higher biofilm-forming capacity: 63.1% (54.0% for E. coli strains) vs. 42.3% for BSI strains (47.7% for E. coli). Strains isolated from UTIs did not present higher mutation frequencies than those from BSIs: contrary to what has been widely described for P. aeruginosa strains, isolated from pulmonary samples in patients suffering from cystic fibrosis, no relationship was found between the hypermutator phenotype in Enterobacteriaceae and the ability to initiate a biofilm.

Virulence-related traits of epidemic Acinetobacter baumannii strains belonging to the international clonal lineages I-III and to the emerging genotypes ST25 and ST78

BackgroundAcinetobacter baumannii is responsible for large epidemics in hospitals, where it can persist for long time on abiotic surfaces. This study investigated some virulence-related traits of epidemic A. baumannii strains assigned to distinct MLST genotypes, including those corresponding to the international clones I-III as well as emerging genotypes responsible for recent epidemics.MethodsGenotyping of bacteria was performed by PFGE analysis and MLST according to the Pasteur’s scheme. Biofilm formation on polystyrene plates was assessed by crystal violet staining; resistance to desiccation was evaluated on glass cover-slips when kept at room-temperature and 31% relative humidity; adherence to and invasion of A549 human alveolar epithelial cells were determined by the analysis of viable bacteria associated with or internalized by A549 human alveolar epithelial cells; Galleria mellonella killing assays were used to analyze the virulence of A. baumannii in vivo.ResultsThe ability to form biofilm was significantly higher for A. baumannnii strains assigned to ST2 (international clone II), ST25 and ST78 compared to other STs. All A. baumannii strains survived on dry surfaces for over 16 days, and strains assigned to ST1 (international clone I) and ST78 survived for up to 89 and 96 days, respectively. Adherence to A549 pneumocytes was higher for strains assigned to ST2, ST25 and ST78 than other genotypes; a positive correlation exists between adherence and biofilm formation. Strains assigned to ST78 also showed significantly higher ability to invade A549 cells. No significant differences in the killing of G. mellonella worms were found among strains.ConclusionsElevated resistance to desiccation, high biofilm-forming capacity on abiotic surfaces and adherence to A549 cells might have favoured the spread and persistence in the hospital environment of A. baumannii strains assigned to the international clones I and II and to the emerging genotypes ST25 and ST78.

The relationship between inhibition of bacterial adhesion to a solid surface by sub-MICs of antibiotics and subsequent development of a biofilm

Many studies have demonstrated that subminimal inhibitory concentrations (sub-MICs) of antibiotics can inhibit initial microbial adherence to medical device surfaces. It has been suggested that, by inhibiting initial adhesion, biofilm formation might be prevented. However, since initial adherence and subsequent biofilm formation may be two distinct phenomena, conclusions regarding the effects of sub-MIC antibiotics on initial adhesion cannot be extrapolated to biofilm formation. In this study, we evaluated the adherence of several clinical isolates of coagulase-negative staphylococci (CoNS) to acrylic and the effect of sub-MICs of vancomycin, cefazolin, dicloxacillin and combinations of these antibiotics on adherence and biofilm formation. Most of the antibiotics used resulted in effective reduction of bacterial adherence to acrylic, in some cases reaching over 70% inhibition of adherence. When strains with a high biofilm-forming capacity were grown in sub-MICs of those antibiotics, there existed combinations of the drugs that significantly inhibited biofilm formation. However, most of the antibiotic combinations that inhibited adherence did not have a profound effect on biofilm formation. When comparing the results of the effect of sub-MIC amounts of antibiotics in inhibiting adherence with their effect on the inhibition of biofilm formation, significant differences were found, mainly when using combinations of antibiotics. In general, the effect on the inhibition of adherence was greater than the effect on inhibiting biofilm formation. These results demonstrate that assays evaluating the inhibition of initial adherence to medical surfaces cannot fully predict the effect on inhibition of biofilm formation.