Biofilm Production Capacity Research Articles

THE PREVALENCE OF (OMPA, CSUE) GENES AMONG BIOFILM PRODUCER ACINETOBACTER BAUMANNII ISOLATES

Acinetobacter baumannii is a significant public health problem because it is capable of forming biofilms that may be responsible for the survival of this pathogen in the hospital environment. The aim of this study is to determine the role of (OmpA, CsuE) genes in biofilm production among A. baumannii isolates. A total of 100 clinical isolates of bacteria were collected from a different sources. All isolates were identified by biochemical test, specific selective media (CHROMagar) and polymerase chain reaction by detection presence of (blaOXA51) gene. only 60 isolates were diagnosed as A. baumannii, and to determine the biofilm production capacity two different methods were used Congo red agar method(CRA) and Microtiter plate method (MTP). MTP method result showed 85% of isolates were able to form biofilms, on the other hand, 33.33% of isolates had a biofilm production ability by showing bright black colonies on CRA. Conventional PCR was used to detect the presence of (OmpA, CsuE) genes and the result showed the prevalence of both genes in all isolates were 97%(58/60). It also shows a strong correlation between the presence of these genes and biofilm formation.

Virulome and phylogenomic profiling of a novel Burkholderia pseudomallei strain from an Indian clinical isolate.

Highly pathogenic Burkholderia pseudomallei is the causative agent of melioidosis, a neglected tropical disease endemic in Southeast Asian tropical region. This bacterium encompasses diverse virulence factors which further undergo dynamic gene-expression flux as it transits through distinct environmental niches within the host which may lead to manifestation of differential clinical symptoms. B. pseudomallei, is classified as a Tier 1 select agent in the United States and regarded as a risk group 3 organism in India with the potential to be used as bioweapon. Considering these facts, it is vital to uncover both physiological and genetic heterogeneity of B. pseudomallei, particularly to identify any novel virulence factors that may contribute to pathogenicity. B. pseudomallei strain CM000113 was isolated from a clinical case in India, characterized it for its physiological, biochemical, and prominently genetic traits through WGS. It has a type 2 morphotype with faster doubling time and high biofilm producing capacity as compared to Pseudomonas aeruginosa. The genome size is 7.3 Mbp and it is phylogenetically close to B. pseudomallei strain Mahidol 1106a and Burkholderia mallei Turkey 2. We observed genetic heterogeneity, as key virulence factors that were identified shows sequence dissimilarity with reference strains. Additionally, presence of genomic islands, harbouring two virulence factors, GmhA and GmhB2, associated with pathogenesis indicates possibility of horizontal gene transfer. These results emphasize the need for an extensive study focusing the genome of B. pseudomallei and its associated heterogeneity, to identify molecular biomarkers aiding to develop point-of-care diagnostic kits for early diagnosis of melioidosis.

Virulence factors, antibiotic susceptibility and sequence type distribution of hospital-associated Clostridioides difficile isolates in Israel, 2020–2022

Biofilm formation and toxin production are some of the virulence factors of Clostridioides difficile (C. difficile), which causes hospital-acquired C. difficile infection (HA-CDI). This work investigated the prevalence and distribution of different strains recovered from HA-CDI patients hospitalized in 4 medical centres across Israel, and characterized strains' virulence factors and antibiotic susceptibility. One-hundred and eighty-eight faecal samples were collected. C. difficile 's toxins were detected by the CerTest Clostridium difficile GDH + Toxin A + B combo card test kit. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Multi-locus sequence typing (MLST) was performed to classify strains. Biofilm production was assessed by crystal violet. Antibiotic susceptibility was determined using Etest. Fidaxomicin susceptibility was tested via agar dilution. Sequence type (ST) 42 was the most (13.8%) common strain. All strains harboured the 2 toxins genes; 6.9% had the binary toxin. Most isolates were susceptible to metronidazole (98.9%) and vancomycin (99.5%). Eleven (5.85%) isolates were fidaxomicin-resistant. Biofilm production capacity was associated with ST (p < 0.001). In conclusion, a broad variety of C. difficile strains circulate in Israel's medical centres. Further studies are needed to explore the differences and their contribution to HA-CDI epidemiology.

In vitro sensitivity of Candida albicans isolates to antifungal agents and the effect of biofilms on the drug resistance rate

Background and objectives: The pathogenic species that is most frequently isolated from fungus infections is Candida albicans. The host's immune system, the severity of the infection, and the antifungal medication of choice all influence the management of these infections. Despite the advancement of novel antifungal medications, epidemiological investigations have demonstrated that resistance to antifungal agents in C. albicans strains is becoming a significant issue. The aims of this study were to (I) determine the prevalence of antifungal resistance in 53 oral Candida albicans isolates and (II) investigate the effect of biofilm production capacity on antifungal resistance rates. Methods: Fifty-three isolates of Candida albicans were evaluated for biofilm production using the Tissue Culture Plate Method (TCPM). The antifungal susceptibility pattern of these isolates for amphotericin B, caspofungin, anidulafungin, voriconazole, itraconazole, ketoconazole, and posaconazole was determined using the E-test. Results: The present study showed strong, moderate, weak, and negative biofilm production in 18.9%, 34.0%, 26.4%, and 20.8% of the C. albicans isolates, respectively. Amphotericin B, anidulafungin, and caspofungin effectively inhibited all of the tested isolates. The resistance rates of C. albicans isolates to fluconazole, itraconazole, ketoconazole, voriconazole, and posaconazole were 35.8%, 20.7%, 37.7%, 13.2%, and 20.7%, respectively. Strong and moderate biofilm-producing isolates displayed higher resistance rates compared to weak and negative biofilm producers. The majority of antifungal treatments showed a significant difference (p &lt;0.05)". Conclusion: Our results suggest that the study area has a higher prevalence of azole-resistant Candida albicans. Thus, there is an urgent need to develop a plan to decrease the overuse and unneeded adverse effects of antifungal medications. Testing for antifungal susceptibility and fungus culture will be helpful in monitoring resistance and providing therapy for patients. It was observed that the development of Candida albicans biofilms was correlated with the drug resistance component of oral Candida albicans isolates.

Effect of electroinductive conditions on biofilm production capacity of some industrially important bacteria

Biofilms are intricate microbial deposits on biotic and abiotic surfaces, with significant medical and biotechnological implications. This study explored biofilm formation by Acetobacter aceti ATCC15973, Pseudomonas aeruginosa ATCC9027, Serratia marcescens ATCC14756, Gluconobacter oxydans ATCC19357, Rhodobacter sphaeroides ATCC17023, and Bacillus subtilis ATCC6633 on wood, glass, steel, PVC, and PET surfaces using qualitative methods. Effects of electrical stimulation (6V, 4.5A), magnetic fields (1000 G), and electromagnetic flux (5 mT) on biofilm formation were assessed via Crystal Violet Binding Assay. G. oxydans ATCC19357 exhibited highest adherence on PVC and wood (2.0145 and 2.402 log cfu/ml, respectively) under electrical stimulation. A. aceti ATCC15973 showed highest adherence on steel, PET, and glass (1.944, 0.9005, and 0.876 log cfu/ml). R. sphaeroides ATCC17023 demonstrated highest adherence on PVC, steel, PET, and glass (1.0895 to 1.7495 log cfu/ml) under magnetic induction; B. subtilis ATCC6633 had highest wood adherence (1.491 log cfu/ml). G. oxydans ATCC19357 showed highest overall adhesion with electromagnetic induction. PVC supported highest biofilm growth (39 %). Biophysical factors varied in enhancing biofilm formation, suggesting potential for bacterial immobilization technologies in bioremediation and industrial fermentation

Genetic Complexity of CC5 Staphylococcus aureus Isolates Associated with Sternal Bursitis in Chickens: Antimicrobial Resistance, Virulence, Plasmids, and Biofilm Formation.

Sternal bursitis, a common inflammatory condition in poultry, poses significant challenges to both animal welfare and public health. This study aimed to investigate the prevalence, antimicrobial resistance, and genetic characteristics of Staphylococcus aureus isolates associated with sternal bursitis in chickens. Ninety-eight samples were collected from affected chickens, and 24 S. aureus isolates were identified. Antimicrobial susceptibility testing revealed resistance to multiple agents, with a notable prevalence of aminoglycoside resistance genes. Whole genome sequencing elucidated the genetic diversity and virulence profiles of the isolates, highlighting the predominance of clonal complex 5 (CC5) strains. Additionally, biofilm formation assays demonstrated moderate biofilm production capacity among the isolates. These findings underscore the importance of vigilant monitoring and targeted interventions to mitigate the impact of sternal bursitis in poultry production systems.

Assessment of Biofilm Forming Capability and Antibiotic Resistance in Proteus mirabilis Colonizing Indwelling Catheter.

&lt;b&gt;Background and Objective:&lt;/b&gt; Urinary tract infections from the use of an indwelling urinary catheter are one of the most common infections caused by &lt;i&gt;Proteus mirabilis&lt;/i&gt;. Due to their biofilm-producing capacity and the increasing antimicrobial resistance in this microorganism, this study aimed to determine the prevalence, biofilm-producing capacity, antimicrobial resistance patterns, multidrug resistance and plasmid mediated resistance of the recovered isolates. &lt;b&gt;Materials and Methods:&lt;/b&gt; A total of 50 urinary samples were collected from May to August, 2018 from patients on indwelling urinary catheters. Using routine microbiological and biochemical methods, 37 &lt;i&gt;P. mirabilis&lt;/i&gt; were isolated. Biofilm forming capability was determined among the isolates using the tube method while antimicrobial susceptibility and plasmid curing were also performed. &lt;b&gt;Results:&lt;/b&gt; All isolates were biofilm producers with 17(46%) being moderate producers while 20(54%) were strong biofilm formers. The study isolates exhibited a high resistance rate to empiric antibiotics, including ceftazidime (75.8%), cefuroxime (54.5%), ampicillin (69.7%) and amoxicillin-clavulanic acid (51.5%). Low resistance was seen in the fluoroquinolones, gentamicin and nitrofurantoin. Plasmid curing experiment revealed that most isolates lost their resistance indicating that resistance was borne on plasmids. Plasmid carriage is likely the reason for the high MDR rate of 56.8% observed. &lt;b&gt;Conclusion:&lt;/b&gt; These findings necessitate the provision of infection control programs which will guide and implement policies.

Determination of the Activity of the fimF Gene and Its N-Terminal Domain Disrupted Mutant on Biofilm Formation and Its Contribution to the Oxidative Stress Response in S. Typhimurium

Fimbriae is an important virulence factor which plays a key role in cell attachment and colonization of the intestinal mucosa during an infection of Salmonella, a pathogen that causes gastroenteritis and systemic infection in humans. In S. Typhimurium, type 1 fimbriae production strengthens the oxidative stress response. This study aimed to determine the effectiveness of the fimF gene and its N-terminal domain on biofilm formation in S. Typhimurium and their contribution to the oxidative stress response. Before the experiments to prove whether the N-terminal domain of the FimF protein is the region that determines the mechanism and function of the fimF gene; only the N-terminal domain of the fimF gene was cloned behind the pBAD promoter. As a result of biofilm experiments on polystyrene surfaces, it was determined that the biofilm production capacity was reduced significantly in mutant strains in terms of fimF and dam genes (p &lt; 0.05). In the oxidative stress response experiment conducted in the presence of hydrogen peroxide (H2O2), it was determined that the mutant strains were more resistant to hydrogen peroxide than the wild-type strain, therefore Salmonella cells perceived the absence of Dam methylase enzyme and FimF protein as a critical internal stress condition and produced strong responses to these stress conditions. As a result of comparative analysis of the N-terminal domain cloned mutant strain with the wild-type, it was proven that the N-terminal domain of the protein in question acts as an adapter protein, due to its close similarities with the wild-type.

DETECTION OF BIOFILM PRODUCTION FOR ORAL BACTERIAL ISOLATES AND IT’S IMPACT ON DENTAL CARIES OCCURANCE

Background and aims: Microorganisms are known to be involved in the formation of biofilms. These biofilms are often seen in the oral cavity, tooth surfaces, prosthesis surfaces, attached oral mucosa, dental implants, etc. These are capable of causing dental caries or gingivitis, and most of them are also known to possess a higher ability to cause dental caries than non-biofilm-producing bacteria. This study was conducted to detect the biofilm formation in bacterial isolates from the oral cavity of patients attaining dental clinics in the Faculty of Dentistry, Sana’a University. In addition, the study examined the association between DMFT, decay, missing teeth due to caries, and filled teeth in the permanent teeth and the degree of bacteria ability to produce biofilm. Materials and Methods: Biofilm production was performed on 294 oral bacteria isolates from 100 patients who visited dental clinics run by Sana'a University's Faculty of Dentistry and private dental clinics. Biofilm-forming oral bacteria were detected by the tissue culture plate (TCA) method. The impact of biofilm production was correlated with the DMFT index of the tested patients. Results: Out of 294 isolates, biofilm formation was seen in 285 isolates (96.9%) by the TCP method. 21.4% of the iosolates showed a weak ability to produce biofilms, 72.4% showed moderate positivity, and only 3.1% showed strong positivity for biofilm production. Most S. aureus strains showed moderate and strong biofilm production (93.8% and 4.2%, respectively); S. mutans had 86.6% of strains with moderate biofilm production and 1.2% with strong biofilm production, while other streptococci had less biofilm production capacity. The DMFT index for the Yemeni patients included in the study was 5.9±2.4; there was a higher mean±SD (6.2±1.9) of DMFT for weak biofilm-producing bacteria with a difference equal to 3.5, 95% CI=1.4-4.2 (p=0.0001); for moderate biofilm-producing bacteria (6.9±2.5, difference=3.5, 95% CI=1.8-5.2, p=0.0001); and for strong-producing biofilms (mean±SD=6.2±2.9, p=0.03). Conclusion: In conclusion, a higher DMFT was observed in biofilm producers than in non-biofilm producers. The prime biofilm producers were S. aureus, S. mutans, and E. coli. Peer Review History: Received 6 December 2023; Revised 30 January 2024; Accepted 26 February; Available online 15 March 2024 Academic Editor: Dr. Asia Selman Abdullah, Pharmacy institute, University of Basrah, Iraq, asia_abdullah65@yahoo.com Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 7.0/10 Reviewers: Ahmad Najib, Universitas Muslim Indonesia, Makassar, Indonesia, ahmad.najib@umi.ac.id Dr. Mohsen Ali Al-Hamzi, Thamar University, Factuality of Dentistry, Yemen. mohsenalhamzi@gmail.com

Characterisation of highly virulent and colistin-resistant ST367-KL1 Klebsiella quasipneumoniae subsp. similipneumoniae Strain

ObjectivesTo elucidate the characteristics of a colistin-resistant and hypervirulent Klebsiella quasipneumoniae subsp. similipneumoniae strain (KP8) using whole genome sequencing and various phenotypic assays. MethodsAntimicrobial susceptibility testing was performed using broth microdilution. Whole genome sequencing and comparative genomics were utilised to elucidate genomic characteristics. Phenotypic assays to evaluate virulence factors included measurements of mucosal viscosity, biofilm production, siderophore production, infection of A549 cells, serum-killing assays, and Galleria mellonella infection models. ResultsWhole-genome sequencing revealed that the strain (KP8) belongs to sequence type 367 (ST367) and capsular type 1 (KL1), and it harbours several virulence genes, including regulator of mucoid phenotype (rmpA/A2), salmochelin (iroBCDN) and aerobactin (iucABCDiutA). Antibiotic susceptibility tests showed that KP8 was resistant to colistin. Genome analysis showed that the colistin resistance of KP8 might be related to amino acid insertions in pmrB (L215_D217, insL) and pagP (M1_S3, insV). Importantly, KP8 demonstrated comparable mucosal viscosity, biofilm production capacity, siderophore production levels to hvKP. Serum-killing experiments, A549 cell infection models, and G. mellonella infection models further indicated that KP8 displayed high virulence, akin to the hypervirulent strain NUTH-K2044. Notably, global genome analysis of the K. quasipneumoniae subsp. similipneumoniae strains highlighted that the ST367 lineage has a higher tendency to carry virulence-associated genes compared to other sequence types. The prevalence of virulence-associated factors concentrated within Chinese ST367 isolates reinforces this observation. ConclusionThese findings further enhance our understanding of the resistance and pathogenicity of ST367 K. quasipneumoniae subsp. similipneumoniae strain and also providing a broader perspective on the global epidemiological landscape.

Inhibitory effect of selenium nanoparticles on the biofilm formation of multidrug-resistant Acinetobacter baumannii

Background/Aim: Treatment of infections caused by biofilm-producing multidrug-resistant (MDR) pathogens represents a huge global problem due to primary antimicrobial multi-resistance enhanced by reduced penetration of antibiotics in the biofilm-embedded bacteria. The aim of this study was to determine the capacity of biofilm production among MDR Acine-tobacter baumannii (A. baumannii) isolates obtained from different clinical specimens and to evaluate the inhibitory effect of selenium nanoparticles (SeNPs) coated with cationic polymer cetyltrimethylammonium bromide (CTAB) on the biofilm formation. Methods: Antimicrobial effect of antibiotics (meropenem, imipenem, gentamicin, amikacin, ciprofloxacin, levofloxacin and trimethoprim-sulfa-methoxazole) was determined by disk-diffusion assay, while sensitivity to colistin was determined with E test. All 60 isolates were tested on biofilm production in microtiter plates with crystal violet dye. Minimal biofilm inhib-itory concentration (MBIC) of SeNPs was tested in order to prevent biofilm formation in microtiter plates. Results: All tested clinical isolates were classified as MDR (n = 60) and extensively drug-resistant (XDR, n = 60). Out of the total 60 isolates, 55 isolates (92 %) showed the ability for biofilm formation, with the majority of them classified as strong (42 %) and moderate (42 %) biofilm producers. MBIC values of SeNPs for 55 biofilm-producing isolates ranged from 0.07 to 1.25 mg/mL. Strong biofilm producers had statistically higher MBIC (0.15 mg/mL) in correlation to other biofilm-producing isolates (0.07 mg/ mL). There was no correlation between invasiveness of isolates with biofilm production and MBIC values. Conclusion: Presented results are very promising and interesting especially in nanotechnology and medical fields, while SeNPs with the addition of cationic surfactant inhibit biofilm formation of MDR A. baumannii clinical isolates.

Associations of motility and auto-aggregation with biofilm-formation capacity levels in Clostridioidesdifficile

Clostridioides difficile (C. difficile) is responsible for one of the most common nosocomial infections worldwide. This work assessed associations between biofilm-formation capacity levels of C. difficile and cell viability, motility, flagella, motility and auto-aggregation in 118 clinical isolates. Biofilm production was assessed by the crystal violet method. Cell viability was determined by BacTiter-Glo™ Microbial Cell Viability Assay and live-imaging microscopy. Expression levels of LuxS, Cwp84, Spo0A, PilA, and FliC were measured by real-time PCR. Motility was visually assessed in agar tubes. Auto-aggregation levels were determined by OD600 measurements. Out of 118 isolates, 66 (56 %) were biofilm producers, with most being strong or moderate producers. Cell viability, motility and auto-aggregation positively correlated with biofilm-production capacity (p = 0.0001, p = 0.036 and p < 0.0001, respectively). Positive associations were found between pilA, fliC and luxS expression levels and biofilm-production capacity (p = 0.04, p = 0.01, p = 0.036, respectively). This is the first report of associations between biofilm-formation capacity and cell viability, pilA, fliC, and luxS gene expression, auto-aggregation and motility. These correlations should be further explored to expand knowledge on the regulation of C. difficile biofilm formation, and pathogenesis, which will have notable implications on treatment options.

Evaluation of bottled drinking water and occurrence of multidrug-resistance and biofilm producing bacteria in Nepal

Health consequences arising from unsafe drinking water and water insecurity lead to increased reliance on usage of bottled water. Biofilm-producing antibiotic-resistant bacteria in bottled water may pose a risk to public health. This study aims to assess the quality of bottled drinking water with a focus on biofilm-producing and drug-resistant coliform. We analyzed 60 bottled water samples of 30 different brands purchased from Kathmandu for physicochemical and microbial analysis. The parameters pH, iron, total coliform count, Escherichia coli count, and fecal coliform count exceeded National Drinking Water Quality Standards, 2022 in 30.00%, 16.67%, 66.67%, 23.33%, and 16.67% of samples, respectively. Water quality index measurement showed 36.67% and 6.67% of bottled water samples were categorized as grade A and grade B indicating excellent and good water quality, respectively. However, 56.67% of bottled water samples fall under grade E meaning unsuitable for drinking. Among 14 coliform isolates, 85.71% and 14.29% were identified as E. coli and Klebsiella spp, respectively. The antibiotic susceptibility testing revealed that 28.57% of the isolates were multidrug-resistant and Gentamicin resistant isolates comprised 71.43%. However, none of the isolates were carbapenem (meropenem) resistant. In this study, 42.87% of the isolates were found biofilm producers with 14.29% each of strong, moderate, and weak biofilm producers. The genetic potential of biofilm-producing capacity of the isolates was assessed by Polymerase Chain Reaction amplification of bcsA and csgD genes. Our results showed that 66.67% and 50.00% of the isolates harbored bcsA and csgD genes, respectively. This study highlights potential public health hazards associated with the consumption of bottled water containing biofilm-producing and drug-resistant bacteria in Nepal.

Lactiplantibacillus plantarum from Unexplored Tunisian Ecological Niches: Antimicrobial Potential, Probiotic and Food Applications.

The continued exploration of the diversity of lactic acid bacteria in little-studied ecological niches represents a fundamental activity to understand the diffusion and biotechnological significance of this heterogeneous class of prokaryotes. In this study, Lactiplantibacillus plantarum (Lpb. plantarum) strains were isolated from Tunisian vegetable sources, including fermented olive and fermented pepper, and from dead locust intestines, which were subsequently evaluated for their antimicrobial activity against foodborne pathogenic bacteria, including Escherichia coli O157:H7 CECT 4267 and Listeria monocytogenes CECT 4031, as well as against some fungi, including Penicillium expansum, Aspergilus niger, and Botrytis cinerea. In addition, their resistance to oro-gastro-intestinal transit, aggregation capabilities, biofilm production capacity, adhesion to human enterocyte-like cells, and cytotoxicity to colorectal adenocarcinoma cell line were determined. Further, adhesion to tomatoes and the biocontrol potential of this model food matrix were analyzed. It was found that all the strains were able to inhibit the indicator growth, mostly through organic acid production. Furthermore, these strains showed promising probiotic traits, including in vitro tolerance to oro-gastrointestinal conditions, and adhesion to abiotic surfaces and Caco-2 cells. Moreover, all tested Lpb. plantarum strains were able to adhere to tomatoes with similar rates (4.0-6.0 LogCFU/g tomato). The co-culture of LAB strains with pathogens on tomatoes showed that Lpb. plantarum could be a good candidate to control pathogen growth. Nonetheless, further studies are needed to guarantee their use as probiotic strains for biocontrol on food matrices.

Investigation of genetic relatedness, antimicrobial resistance, biofilm formation, biofilm-related virulence genes and integron-related genes of Stenotrophomonas maltophilia isolates obtained from bovine milk samples with mastitis

Treatment of infections caused by opportunistic pathogenic Stenotrophomonas maltophilia is complicated by the bacterium's ability to produce biofilms and high antibiotic resistance. This study aimed to investigate the prevalence of genetic relatedness, antimicrobial resistance, biofilm formation, biofilm genes associated with virulence and integron genes among isolates of S. maltophilia recovered from bovine milk with subclinical mastitis. In this study, bacterial identification was performed using conventional methods. While using the smeT gene-based Polymerase Chain Reaction (PCR) to confirm the species-level identification of isolates; PCR was also used to detect virulence and integron genes, too. The quantitative Microplate Test (MP) method was used to determine the phenotypic biofilm production capacity of the isolates. The resistance patterns of the isolates against 9 antibiotics belonging to 9 antimicrobial families were examined using the disk diffusion method. Isolates resistant to at least three drug classes from various antimicrobial drug classes were defined as multi-drug resistant (MDR). The genetic linkage of S. maltophilia isolates was investigated by Enterobacterial Repetitive Intragenic Consensus (ERIC) PCR. The Chi-Square (χ2) test was used to compare the relationship between the biofilm-forming capacity of the isolates with the prevalence of biofilm-associated virulence genes and integron genes with MDR. In the study, a total of 312 milk samples with subclinical mastitis were taken from 27 farms. Ten isolates from five farms were phenotypically and genotypically identified as S. maltophilia. All isolates were resistant to cefepime and imipenem. While 70% of the isolates were MDR; 80% carried one of the integron genes. By the MP test, the phenotypically biofilm-forming capacity identified in isolates was detected at 80%. The prevalence of the studied virulence genes was rpfF 60%, rmlA 70%, spgM and smf1 80%. There was no significant relationship between the biofilm-forming capacity of the isolates with the prevalence of biofilm-associated virulence genes and MDR with integron genes. S. maltophilia isolates were detected simply and quickly, using PCR based on the smeT gene, from bovine milk samples for the first time in Turkey. In the UPGMA analysis performed in the PyElph 1.4 program, a total of 5 genotypes were found, 2 single and 3 multiple according to 18% similarity coefficient. ERIC-PCR can be useful in identifying S. maltophilia isolates with epidemic potential.

Photodynamic Therapy and Its Synergism with Melittin Against Drug-Resistant Acinetobacter baumannii Isolates with High Biofilm Formation Ability.

Drug-resistant biofilm producer A. baumannii isolates are a global concern that warns researchers about the development of new treatments. This study was designed to analyze the effect of photodynamic therapy (PDT) as monotherapy and associated with melittin on multidrug-resistant A. baumannii isolates. Sub-lethal doses of photosensitizer, LED, and PDT were determined. The PDT effect on the biofilm and expression of biofilm-associated genes was evaluated by scanning electron microscopy and quantitative real-time PCR (qRT-PCR) methods, respectively. The synergistic effect of PDT and melittin on the survival of MDR/XDR strong biofilm producer isolates was evaluated by checkerboard assay. Survival rates were significantly decreased at the lowest concentration of 12.5-50μg/ml in 4min at an energy density of 93.75J/cm2 (P < 0.05). The optimized PDT method had a bactericidal effect against all tested groups, and the mean expression levels of csu, abaI, bap, and ompA genes in the strong biofilm producers were decreased significantly compared to the control group. The combined effect of LED and melittin successfully reduced the MDR/XDR A. baumannii strong biofilm producers' growth from 3.1 logs. MB-mediated aPDT and combined treatment of PDT with melittin, which has been investigated for the first time in this study, can be an efficient strategy against MDR/XDR A. baumannii isolates with strong biofilm production capacity.

Biofilm Formation and Aspartyl Proteinase Activity and Their Association with Azole Resistance Among Candida albicans Causing Vulvovaginal Candidiasis, Egypt.

Candida albicans (C. albicans) is a major cause of vulvovaginal candidiasis (VVC), a condition that is commonly treated with azole agents. Biofilm formation and aspartyl proteinase production are important virulence factors that could be linked to azole resistance in C. albicans impeding therapy. To find out the association of both factors with azole resistance among C. albicans isolated from VVC cases in Egyptian nonpregnant women of childbearing age. In a cross-sectional study, C. albicans was isolated from nonpregnant females diagnosed clinically as having VVC during a 1-year study period. Susceptibility to azole agents was tested using the disc diffusion method. Biofilm formation and aspartyl proteinase production were assessed phenotypically. Additionally, two biofilm-related genes (ALS1 and HWP1) and three proteinase genes (SAP2, SAP4, and SAP6) were screened for using polymerase chain reaction (PCR). Among 204 C. albicans isolates, azole resistance ratios were as follows: voriconazole (30.4%), itraconazole (17.6%), fluconazole (11.3%) and econazole (6.4%). Biofilm-producing capacity was detected in 63.2% of isolates, and 63.2% were proteinase producers. The frequencies of ALS1 and HWP1 were 69.6% and 74.5%, respectively, while SAP2, SAP4, and SAP6 were 69.2%, 88.7%, and 64.7%, respectively. Biofilm formation was significantly associated with azole resistance (P < 0.001 for each tested azole agent) as was proteinase production (P < 0.001 for fluconazole, voriconazole, and econazole resistance and P = 0.047 for itraconazole). Among nonpregnant Egyptian women of childbearing age, azole resistance in C. albicans causing VVC is significantly associated with biofilm formation and proteinase production. The development of new therapeutic agents that can target these factors is warranted.

Extensive Drug Resistance of Strong Biofilm-Producing Acinetobacter baumannii Strains Isolated from Infections and Colonization Hospitalized Patients in Southern Poland.

Acinetobacter baumannii (AB) is a bacterium that causes infections, particularly in immunocompromised patients. Treatment is challenging due to biofilm formation by AB strains, which hinders antibiotic effectiveness and promotes drug resistance. The aim of our study was to analyze the biofilm-producing capacity of AB isolates from various forms of infections in relation to biofilm-related genes and their drug resistance. We tested one hundred isolates for biofilm formation using the crystal violet microplate method. Drug resistance analyses were performed based on EUCAST and CLSI guidelines, and biofilm genes were detected using PCR. All tested strains were found to form biofilms, with 50% being ICU strains and 72% classified as strong biofilm producers. Among these, 87% were extensively drug-resistant (XDR) and 2% were extra-extensively drug-resistant (E-XDR). The most common gene set was bap, bfmS, csuE, and ompA, found in 57% of all isolates. Our research shows that, regardless of the form of infection, biofilm-forming strains can be expected among AB isolates. The emergence of E-XDR and XDR strains among non-ICU infections highlights the necessity for the rational use of antibiotics to stop or limit the further acquisition of drug resistance by A. baumannii.

Capacity of biofilm production by filamentous fungi isolated from the uterus of susceptible mares

Fungi are opportunistic microorganisms that adhere to surfaces. Their intrauterine growth is influenced by impaired immunity, indiscriminate use of antibiotics, and poor vulvar conformation. Many publications use yeast research models, but only some report biofilm production by filamentous fungi. This work aimed to biofilm capacity from isolates of filamentous fungi found in uterine samples from susceptible mares. Uterine saline lavage samples from 41 mares with a history of subfertility were collected and sent to LABRAPE-UFRPE, Brazil. Samples were cultured on Sabouraud Dextrose Agar plates with 10% chloramphenicol and incubated at 25°C for up to 15 days, inspecting every 24 hours. After development of the colonies, identification used the macro and micromorphological aspects of the somatic and reproductive structures, comparing them with the literature. To induce biofilm production, isolates were re-cultured on Sabouraud dextrose agar at 37°C for 72 h. Conidia were collected by washing the surface of the cultures using 5mL PBS, pH 7.2, supplemented with 0.025% (v/v) Tween 20. Subsequently, conidia were counted in a Neubauer chamber, and the inoculum was adjusted to the concentration of 1 × 105 cells in RPMI 1640and buffered to pH 7.0. Biofilms were produced in 96-well microtiter plates by adding 200µL of standardized cell suspension in MOPS-RPMI 1640 buffer in each well, at each determined period (24, 48, 72, and 96h) then incubated at 37°C. At each time interval, biofilms were washed three times with PBS pH 7.2 then quantified using crystal violet technique (absorbance 570nm). Of the samples, 12.2% were positive for fungal endometritis, with Cladosporium spp. isolated in 60% of the positive samples, Trichosporon spp. and Penicillium spp. in 20% of the positive samples each. After induction of biofilm production, fungi of the genus Cladosporium spp. and Penicillium spp. showed a high biofilm production capacity, with a growth peak at 48 hours after incubation and a gradual decrease in this production in the subsequent hours. Cladosporium spp. showed low biofilm production until 96h of incubation, and Penicillium spp zeroed its formation simultaneously. In conclusion filamentous fungi isolated from the uterus of mares have a greater biofilm production capacity after 48 hours incubation, which tends to decrease over time but can be maintained for up to 96 hours, depending on the gender involved in the infection, as well as the quantity and quality of the substrate available.

Characterization of the Biofilms Formed by Histamine-Producing Lentilactobacillus parabuchneri Strains in the Dairy Environment

Lentilactobacillus parabuchneri, a lactic acid bacterium, is largely responsible for the production and accumulation of histamine, a toxic biogenic amine, in cheese. L. parabuchneri strains can form biofilms on the surface of industry equipment. Since they are resistant to cleaning and disinfection, they may act as reservoirs of histamine-producing contaminants in cheese. The aim of this study was to investigate the biofilm-producing capacity of L. parabuchneri strains. Using the crystal violet technique, the strains were first categorized as weak, moderate or strong biofilm producers. Analysis of their biofilm matrices revealed them to be mainly composed of proteins. Two strains of each category were then selected to analyze the influence on the biofilm-forming capacity of temperature, pH, carbon source, NaCl concentration and surface material (i.e., focusing on those used in the dairy industry). In general, low temperature (8 °C), high NaCl concentrations (2-3% w/v) and neutral pH (pH 6) prevented biofilm formation. All strains were found to adhere easily to beech wood. These findings increase knowledge of the biofilm-forming capacity of histamine-producing L. parabuchneri strains and how their formation may be prevented for improving food safety.