Microtiter Plate Method Research Articles

Assessment of Antibiotic Resistance Among Isolates of Klebsiella spp. and Raoultella spp. in Wildlife and Their Environment from Portugal: A Positive Epidemiologic Outcome.

One of the significant challenges facing modern medicine is the rising rate of antibiotic resistance, which impacts public health, animal health, and environmental preservation. Evaluating antibiotic resistance in wildlife and their environments is crucial, as it offers essential insights into the dynamics of resistance patterns and promotes strategies for monitoring, prevention, and intervention. Klebsiella and Raoultella genera isolates were recovered from fecal samples of wild animals and environmental samples using media without antibiotic supplementation. Antibiograms were performed for 15 antibiotics to determine the phenotypic resistance profile in these isolates. Extended-spectrum β-lactamase (ESBL) production was tested by the double-disc synergy test, and one ESBL-producing K. pneumoniae isolate was screened by PCR and whole-genome sequencing. Biofilm production was analyzed using the microtiter plate method. A total of 23 Klebsiella spp. and 3 Raoultella spp. isolates were obtained from 312 fecal samples from wild animals, 9 Klebsiella spp. and 4 Raoultella spp. isolates were obtained from 18 river and stream water samples, and 4 Klebsiella spp. and 3 Raoultella spp. isolates from 48 soil samples. Regarding antibiotic resistance, only one isolate of K. pneumoniae from soil samples was an ESBL-producer and showed resistance to six antibiotics. This isolate harbored multiple β-lactams genes (blaCTX-M-15, blaTEM-1, blaSHV-28, and blaOXA-1), as well as genes of resistance to quinolones, sulfonamides, tetracycline, aminoglycosides, and chloramphenicol, and belonged to the lineage ST307. Most of the Klebsiella spp. and Raoultella spp. isolates were biofilm producers (except for one Klebsiella isolate), and 45.6% were weak biofilm producers, with the remaining being moderate to strong biofilm producers. We can conclude that antibiotic resistance is not widespread in these environment-associated isolates, which is a positive epidemiological outcome. However, identifying a single ESBL-K. pneumoniae isolate should serve as a warning of potential hotspots of resistance emergence.

Detection of Some Virulence Factors from Candida spp. Isolated from Vaginal and Oral Candidiasis in Iraqi Patients.

Candidiasis can be present as a cutaneous, mucosal, or deep-seated organ infection, which is caused by more than 20 types of Candida spp., with C. albicans being the most common. Hence, this work aimed to estimate some virulence factors, including phospholipase and biofilm formation, in some Candida spp. A total of eighty-six specimens were collected from patients with oral and vaginal candidiasis and subjected into different examinations, including cultural characteristic (on Sabouraud Dextrose Agar SDA and chromogenic Candida agar) and microscopic examination and germ tube formation (GT)to isolate Candida spp. In addition, the egg-yolk agar plate method was used to determine the extracellular phospholipase production, and the microtiter plate method was used to determine biofilm formation of Candida spp. Vitek Compact equipment was used to identify the highest phospholipase and biofilm-producers of Candida spp. As a result of all examinations, 58.1% (n = 50/86) of isolates of Candida spp. were obtained, including 26.7% (n = 23/86) isolates of Candida spp. from oral cavity and 31.3% (n = 27/86) isolates of Candida spp. from vaginal cavity. These isolates included 58% (n = 29/50) C. albicans, 10% (n = 5/50) C. glabrata, 6% (n = 3/50) C. parasilosis, 6% (n = 3/50) C. krusei, 6% (n = 3/50) C. lusitaniae, 6% (n = 3/50) C. kefyr, 6% (n = 3/50) C. tropicalis, and 2% (n = 1/50) C. ciferrii. To quantify extracellular phospholipase production, the egg-yolk agar plate method was utilized. The results indicated that the majority of isolates (n = 33; 66%) were phospholipase-strong producers, 18% (n = 9) of isolates were phospholipase-moderate producers, 5% (n = 10) were phospholipase-weak producers, and 6% (n = 3) were non-phospholipase producers. Microtiter plate method was utilized to estimate formation of biofilm by Candida spp. obtained from vaginal and oral cavities. The majority of Candida spp. isolates (n = 32; 64%) were biofilm-strong producers, followed by 30% (n=15) moderate-biofilm producers and 6% (n = 3) weak-biofilm producers. The results of VITEK 2 system indicated that the probability of C. albicans, C. krusei, C. kefyr, C. tropicalis, C. lusitaniae, C. glabrata, and C. ciferrii was 98, 95, 94, 91, 85, 93, and 85 %, respectively. Candida albicans was the most frequent isolate among all isolates.

WAR IN THE MIDDLE EAR: MICROBIOLOGY OF CHRONIC SUPPURATIVE OTITIS MEDIA WITH SPECIAL REFERENCE TO ANAEROBES AND ITS ANTIMICROBIAL SUSCEPTIBILITY PATTERN: OPTIMIZING ANTIMICROBIAL THERAPY IN A TERTIARY CARE HOSPITAL OF RURAL INDIA

Objective: To isolate etiological organisms of Chronic Suppurative Otitis Media (CSOM), study the antimicrobial susceptibility pattern, study the risk factors and associated co-morbid conditions with CSOM, and detect biofilm production in isolated bacteria. Methods: An ear discharge specimen was obtained from the diseased ear of the patient, using three separate sterilized swabs. One of the swabs was used for Gram staining and Aerobic culture. Antibiotic susceptibility testing was done by the Kirby-Bauer standard disc diffusion method all the g-negative isolates were screened for Extended-Spectrum Beta-Lactamase (ESBL) production and Amp C ß-lactamase production. Methicillin-Resistant Staphylococcus Aureus (MRSA) screening was done using a Cefoxitin disc. Biofilm production was done using the Tube method and Microtiter plate method. Anaerobic culture was inoculated into Robertson’s Cooked Meat (RCM) broth. Potassium hydroxide (KOH) mount was done using 10% KOH for the presence of budding yeast cells, fungal hyphae, and spores. Tubes showing positive cultures were examined by LactoPhenol Cotton Blue (LPCB) mount. Final Candida speciation and Antifungal susceptibility testing was done using VITEK-2. Results: A total of 500 patients were included in the study, with 62.8% being males. The most common organism isolated was Pseudomonas aeruginosa. Fungi accounted for 30 isolates (5%) and anaerobes were 5 isolates (1%). Gram-negative bacteria were found to be most sensitive to Piperacillin-tazobactam (96%) while the highest resistance was noted to Amoxicillin-clavulanate (65%). 100% of Staphylococcus aureus isolates were sensitive to Vancomycin, Linezolid, and Teichoplanin.13% of S. aureus were found to be Cefoxitin resistant (MRSA). The highest resistance in S. aureus isolates was noted to Ciprofloxacin (65%).13% of isolates of Gram-negative bacteria had ESBL production while Amp C β-Lactamase production was noted in 9% of isolates. Out of 186 isolates tested for biofilm production, 149 isolates (80%) showed biofilm production. A total of 30 fungal isolates were obtained in culture. Aspergillus spp. Accounted for 66.6% of fungal isolates while Candida spp. accounted for 33.3% of fungal isolates. The sensitivity of Candida isolates was tested to Fluconazole, Itraconazole, Voriconazole, and Flucytosine and they were found to be 100% sensitive to all the antifungals tested. Conclusion: CSOM often has polymicrobial pathology and creates a war in the middle ear environment, augmented by mixed aerobic and anaerobic organisms and synergism between aerobic and anaerobic organisms. A high rate of Clindamycin resistance was seen in anaerobe which is noteworthy because this is a frequently utilized antimicrobial for treating anaerobic infections.

A Study of Bacteriological Profiles, Biofilm Production, and Antibiotic Susceptibility among Isolates of Chronic Suppurative Otitis Media at a Tertiary Care Hospital

ABSTRACT Background: Chronic suppurative otitis media (CSOM) is defined as chronic inflammation of the middle ear and mastoid mucosa. Biofilms act as non-selective physical barriers that obstruct antibiotic diffusion and hinder immune responses facilitating chronic bacterial infections and antibiotic resistance. Aims and Objectives: To study biofilm production among bacteria causing CSOM and their antibiotic resistance. Materials and Methods: This hospital-based descriptive type of observational study was done in the ENT department and Department of Microbiology. Ear discharge samples from 100 clinically diagnosed CSOM patients were collected and processed. Bacterial isolates were identified, and drug susceptibility testing was done using the Kirby–Bauer disc diffusion method. Biofilm production was detected by the microtiter plate method. Results: Among 100 patients included in the study, the most predominant isolate was Staphylococcus aureus (44.0%), followed by Pseudomonas aeruginosa (36.0%). Biofilm production was detected in 59.0% of isolates. Biofilm production was observed in 97.7% S. aureus isolates. Among Gram-negative isolates, biofilm production was observed in 40.0% of Klebsiella spp., 36.0% P. aeruginosa, and 22.2% E. coli. All isolates of S. aureus were susceptible to vancomycin, tigecycline, and linezolid (100%). Methicillin resistance was found in 25.6% S. aureus isolates. A susceptibility of 100% was observed with colistin and piperacillin-tazobactam among P. aeruginosa isolates. Conclusion: Ear discharge should be sent for culture and antibiotic susceptibility testing before initiation of empirical antibiotic treatment. Biofilm production is frequently associated with multi-drug resistance, so it should be tested routinely in laboratories. This will help in effective management of CSOM, prevent development of complications, and thereby prevent deafness.

Antibacterial, antibiofilm, and gene expression assessment of ajwain (Trachyspermum ammi) essential oil on drug-resistant gastrointestinal pathogens and its combination effect with ampicillin.

Essential oils are natural substances used as therapeutic agents and food preservatives to inhibit harmful microorganisms. This study aimed to assess the synergistic effect of Trachyspermum ammi essential oil and ampicillin on antibiotic-resistant gastrointestinal pathogens, including Escherichia coli, Enterococcus faecalis, Shigella flexneri, and Salmonella serotype Typhimurium. Using gas chromatography-mass spectrometry (GC-MS), the main components of T. ammi essential oil were identified as thymol, gamma terpenes, and cymene. The antibacterial and antibiofilm properties were evaluated by minimum inhibitory concentration (MIC), disk diffusion, and microtiter plate methods, revealing MIC values of 2, 1, 4, and 4mg ml-1 for E. coli, E. faecalis, S. flexneri, and S. Typhimurium, respectively, and inhibition zones between 10 and 14mm. Pathogens were examined for their biofilm-related virulence genes, including aggR, esp, icsA, and fliC, using real-time polymerase chain reaction (RT-PCR) in E. coli, E. faecalis, S. flexneri, and S. Typhimurium, respectively. The methyl thiazole tetrazolium (MTT) assay was used to evaluate the essential oil's effect on the viability of human embryonic kidney 293 (HEK293) cells, which showed cell viability of over 80%. The combination of T. ammi oil and ampicillin demonstrated a synergistic effect, and biofilm formation was inhibited. E. faecalis exhibited the greatest sensitivity, while S. flexneri exhibited the lowest sensitivity.

Effects of Phenolic Plant Extracts on Biofilm Formation by Klebsiella pneumoniae Isolated from Urinary Tract Infections

Ten isolates of Klebsiella pneumoniae, seven isolates of Pseudomonas aeruginosa and nine isolates of Staphylococcus aureus, were obtained from 100 urine samples collected from Baghdad hospitals. All isolates were identified biochemically and confirmed by using VITEK 2 and were then tested for their susceptibility towards 6 antibiotics and for phenolic extracts of Thymus vulgaris and Cinnamomum cassia. All bacteria were greatly affected by T. vulgaris, especially K. pneumoniae. Viable count was performed, it was noted that the number of bacterial cells reduced from 1×108 CFU to 1.2× 103, 2×105 and 1.8×106CFU of K. pneumoniae, P. aeruginosa and S. aureus respectively. While C. cassiahad a slight effect on them. K. pneumoniae isolates which were affected by phenolic extract more than the other bacteria under study and at the same time were resistant to more than one type of tested antibiotics. These isolates were taken to detect their ability to form biofilm by using Congo red as screening method for it. The results showed that all isolates produced biofilms. Also, by using microtiter plate method, the results confirmed that all isolates produced biofilm where 7 isolates were strong biofilm producers and 3 were moderate. The strongest isolate was taken to study the effect of T. vulgaris and C. cassia phenolic extract on its biofilm formation by using microtiter plate method with two concentrations (20 and 40 ml/L). The results showed that biofilm reduction was 45% and 73% for T. vulgaris and that for C. cassiait was 15% and 20% after using 20 and 40 ml/L respectively.

Biofilm Formation and Plasmid-Mediated Quinolone Resistance Genes at Varying Quinolone Inhibitory Concentrations in Quinolone-Resistant Bacteria Superinfecting COVID-19 Inpatients.

The likelihood of antimicrobial failure in COVID-19 patients with bacterial superinfection arises from both phenotypic (biofilms) and genotypic mechanisms. This cross-sectional study aimed to determine the inhibitory concentrations of quinolones-nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, and levofloxacin-in biofilm formers (minimum biofilm inhibitory concentration [MBIC]) and nonformers (minimum inhibitory concentration [MIC]) and correlate inhibitory concentrations with plasmid-mediated quinolone resistance (PMQR) genes in quinolone-resistant bacteria isolated from COVID-19 inpatients. Quinolone-resistant bacteria (n = 193), verified through disc diffusion, were tested for quinolone inhibitory concentrations using broth microdilution and biofilm formation using microtiter plate methods. The polymerase chain reaction was used to detect PMQR genes. Study variables were analyzed using SPSS v.17.0, with a significance level set at P <0.05. MIC-to-MBIC median fold increases for ciprofloxacin, ofloxacin, and levofloxacin were 128 (2-8,192), 64 (4-1,024), and 32 (4-512) in gram-positive cocci (GPC, n = 43), respectively, whereas they were 32 (4-8,192), 32 (4-2,048), and 16 (2-1,024) in fermentative gram-negative bacilli (F-GNB, n = 126) and 16 (4-4,096), 64 (2-64), and 16 (8-512) in nonfermentative gram-negative bacilli (NF-GNB, n = 24). In biofilm-forming F-GNB and NF-GNB, qnrB (10/32 versus 3/10), aac(6')-Ib-cr (10/32 versus 4/10), and qnrS (9/32 versus 0/10) genes were detected. A 32-fold median increase in the MIC-to-MBIC of ciprofloxacin was significantly (P <0.05) associated with qnrA in F-GNB and qnrS in NF-GNB. Biofilms formed by F-GNB and NF-GNB were significantly associated with the aac(6')-Ib-cr and qnrS genes, respectively. Nearly one-third of the superinfecting bacteria in COVID-19 patients formed biofilms and had at least one PMQR gene, thus increasing the need for quinolones at higher inhibitory concentrations.

The antimicrobial and antibiofilm effects of gentamicin, imipenem, and fucoidan combinations against dual-species biofilms of Staphylococcus aureus and Acinetobacter baumannii isolated from diabetic foot ulcers

IntroductionDiabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia due to impaired insulin production or utilization, leading to severe health complications. Diabetic foot ulcers (DFUs) represent a major complication, often exacerbated by polymicrobial infections involving Staphylococcus aureus and Acinetobacter baumannii. These pathogens, notorious for their resistance to antibiotics, complicate treatment efforts, especially due to biofilm formation, which enhances bacterial survival and resistance. This study explores the synergistic effects of combining gentamicin, imipenem, and fucoidan, a sulfated polysaccharide with antimicrobial properties, against both planktonic and biofilm forms of S. aureus and A. baumannii.MethodsIsolates of S. aureus and A. baumannii were collected from DFUs and genetically confirmed. Methicillin resistance in S. aureus was identified through disk diffusion and PCR. Biofilm formation, including dual-species biofilms, was analyzed using the microtiter plate method. The antimicrobial efficacy of gentamicin, imipenem, and fucoidan was assessed by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC). Synergistic interactions were evaluated using the fractional inhibitory concentration index (FICi) and fractional bactericidal concentration index (FBCi). The expression of biofilm-associated genes (icaA in S. aureus and bap in A. baumannii) was analyzed, and the cytotoxicity of fucoidan was assessed.ResultsThe study revealed that 77.4% of S. aureus and all A. baumannii isolates showed multidrug resistance. Among 837 tested conditions for dual-species biofilm formation, 72 resulted in strong biofilm formation and 67 in moderate biofilm formation. The geometric mean MIC values for gentamicin were 12.2 µg/mL for S. aureus, 22.62 µg/mL for A. baumannii, and 5.87 µg/mL for their co-culture; for imipenem, they were 19.84, 9.18, and 3.70 µg/mL, respectively, and for fucoidan, 48.50, 31.20, and 19.65 µg/mL, respectively. The MBC values for gentamicin were 119.42, 128, and 11.75 µg/mL; for imipenem, they were 48.50, 14.92, and 8 µg/mL; and for fucoidan, they were 88.37, 62.62, and 42.48 µg/mL. The MBIC values were 55.71, 119.42, and 18.66 µg/mL for gentamicin; 68.59, 48.50, and 25.39 µg/mL for imipenem; and 153.89, 101.49, and 53.53 µg/mL for fucoidan. The MBEC values were 315.17, 362.03, and 59.25 µg/mL for gentamicin; 207.93, 157.58, and 74.65 µg/mL for imipenem; and 353.55, 189.46, and 99.19 µg/mL for fucoidan. When cultured in planktonic form, the geometric mean FICi and FBCi values indicated additive effects, while co-culture showed FICi values of ≤ 0.5, suggesting a synergistic interaction. Treatment with gentamicin and fucoidan led to significant downregulation of the icaA and bap genes in both single-species and dual-species biofilms of S. aureus and A. baumannii. The reductions in gene expression were more pronounced in dual-species biofilms compared to single-species biofilms. Additionally, treatment with imipenem and fucoidan also resulted in significant downregulation of these genes in both biofilm types. Cytotoxicity assessments indicated that higher concentrations of fucoidan were toxic, yet no harmful effects were noted at the optimal synergistic concentrations used with antibiotics.ConclusionIn our investigation, we found that combining gentamicin, imipenem, and fucoidan had a synergistic effect on dual-species biofilms of S. aureus and A. baumannii, suggesting potential benefits for treating such infections effectively. This underscores the importance of understanding microbial interactions, antibiotic susceptibility, and biofilm formation in DFUs.

Antibacterial Activity of Silver Nanoparticles Prepared from Camellia Sinensis Extracts in Multi-Drug Resistant Staphylococcus Aureus.

The nano-method has been used as a technique for creating novel, non-traditional antimicrobial agents. This effective method for treating infectious diseases has many advantages over conventional antibiotics, including increased efficacy against species that have developed drug resistance, and the ability to circumvent the development of resistance that disrupts a number of biological pathways. As a result, the objective of this study was to synthesize and characterize silver nanoparticles using phenolic compounds obtained from Camellia sinensis . The nanoparticles were then used as antibacterial agents on the multidrug resistant Staphylococcus aureus, as well as, biofilm formation mechanism were also investigated. Ten isolates of Staphylococcus aureus were acquired from the labs of the University of Baghdad's Genetic Engineering and Biotechnology Institute. The VITEK-2 system was used to confirm the diagnosis. Aqueous and methanolic extracts of Camellia sinensis leaves were used to create silver nanoparticles and obtain CAgNPs, which were then characterized using Atomic Fluorescence Microscopy (AFM), X-ray diffractometer (XRD), and Zeta potential analyzers. The extracts were put through a series of assays, including High-performance liquid chromatography (HPLC), antibacterial activity assessments, and the microtiter plate method to determine the lowest inhibitory concentration (MIC) and antibiofilm formation. Both aqueous and methanolic extracts containing silver nanoparticles included spherical nanoparticles that may be single or combined. The HPLC results showed the presence of two phenolic compounds (gallic acid and caffeine) by comparing their retention durations to those of the reference compounds. The results of the antibacterial activity of (CAgNPs) showed that the methanolic (CAgNPs) extract was more effective than the aqueous (CAgNPs) extract, producing inhibitory zones of 15.67 ± 0.58 and 20.33 ± 0.58 mm, at 375 and 750 ppm respectively, when compared to the aqueous (CAgNPs) extract, which produced inhibitory zones of 12.33 ± 0.58 and 15.67 ± 0.58 mm, respectively. The MIC result also showed that the CAgNPs methanolic extract was more effective than the CAgNPs aqueous extract. The MIC of the CAgNPs methanolic extract on S. aureus isolates were 11.718 and 23.43 µg/ml, while the MIC of the CAgNPs aqueous extract on all S. aureus isolates were 46.87 µg/ml except isolate No. 3 and 6 which was 11.718 and 93.75 µg/ml respectively. Additionally, The anti-biofilm in S. aureus was increased when CAgNPs methanolic extract were used compared with the CAgNPs aqueous extract, the CAgNPs methanolic extract inhibited 80%, 90% and 100% of the biofilm formation of S. aureus in 23.43, 46.87 and 93.75 µg/ml respectively, while the anti-biofilm activity of the CAgNPs aqueous extract on S. aureus isolates was 80% and 100% of the biofilm formation in 46.87 and 93.75 µg/ml respectively. The methanolic and aqueous leaves extracts from Camellia sinensis are a successful method for producing CAgNPs. The synthesized CAgNPs also have significant antibacterial activity against S. aureus, depending on the concentrations, and inhibit S. aureus biofilm formation.

Phenotypic, Molecular Identification and Virulence Assessment of Cronobacter spp Isolated from Clinical Samples of Children Under Two Years in Mosul City/Iraq

Cronobacter spp. is known to be a foodborne causative agent for a variety of diseases in both humans and animals. This study focused on isolating Cronobacter species from 150 clinical samples of children under two years (60 from blood and stool samples, 30 from CSF samples) collected from Ibn al Atheer and Al Khanssa Hospitals. The phenotypic identification of bacterial isolates were performed through culture, microscopic, and biochemical examinations and vitek-2 system. The results revealed a mixture of bacteria in 6.5% of the clinical specimens. The identity of isolates was 99% using the vitek-2 system for identifying Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, and Cronobacter pulveris. Out of 150 specimens there were 8 (5.33 %) of specimens gave positive for Cronobacter spp which included: C. sakazaki 2/60 blood specimens (3.3%), C. sakazaki 2/60 stool specimens (3.3%), C. malonaticus 2/60 stool specimens (3.3%), 1/60 C. muytjensii and C. pulveris 1/60 (3.3%). The eight isolates phenotypically identified as Cronobacter were confirmed at the molecular level through 16S rRNA sequencing and submitted to NCBI under the accession numbers (OR825874, OR825875, PP126443, PP126444, PP126445, PP126455). The virulence profile of these isolates showed that 7/8 (87.5%) of Cronobacter strains exhibited haemolysin activity, 5/8 strains (62.5%) were able to produce the protease enzyme and 2/8 (25%) of Cronobacter strains were positive for lipase and lecithinase. All strains lacked the ability to produce a slime layer. The results also showed the ability of Cronobacter strains to produce biofilm by using the tube method and microtiter plate method but with different levels.

Biofilm formation and antibiogram profile of bacteria from infected wounds in a general hospital in southern Ethiopia

Biofilm-producing bacteria associated with wound infections exhibit exceptional drug resistance, leading to an escalation in morbidity, worse clinical outcomes (including delay in the healing process), and an increase in health care cost, burdening the whole system. This study is an attempt to estimate the prevalence and the relationship between the biofilm-forming capacity and multi-drug resistance of wound bacterial isolates. The findings intended to help clinicians, healthcare providers and program planners and to formulate an evidence-based decision-making process, especially in resource-limited healthcare settings. This study was done to assess the prevalence of bacterial infections in wounds and the antibiogram and biofilm-forming capacity of those bacteria in patients with clinical signs and symptoms, attending a General Hospital in southern Ethiopia. A cross-sectional study was performed in Arba Minch General Hospital from June to November 2021. The study participants comprised 201 patients with clinically infected wounds. Demographic and clinical data were gathered via a structured questionnaire. Specimens from wounds were taken from each participant and inoculated onto a series of culture media, namely MacConkey agar, mannitol salt agar, and blood agar, and different species were identified using a number of biochemical tests. Antimicrobial susceptibility tests were performed by means of the Kirby-Bauer disc diffusion technique following the guidelines of the Clinical and Laboratory Standards Institute. A micro-titer plate method was employed to detect the extent of biofilm formation. Bivariable and multivariable logistic regression models were applied to analyse the association between dependent and independent variables, and P values ≤ 0.05 were considered as statistically significant. Data analyses were done with Statistical Package for the Social Sciences version 25. Out of the 201 clinically infected wounds, 165 were found culture-positive with an overall prevalence of 82% (95% CI: 75.9–86.9). In total, 188 bacteria were recovered; 53.1% of them were Gram-positive cocci. The often-isolated bacterial species were Staphylococcus aureus, 38.3% (n = 72), and Pseudomonas aeruginosa, 16.4% (n = 31). The Gram-positive isolates showed considerable resistance against penicillin, 70%, and somewhat strong resistance against tetracycline, 57.7%. Gram-negative isolates showed severe resistance to ampicillin, 80.68%. The overall multi-drug resistance (MDR) among isolates was 48.4%. Extended beta-lactamase (ESBL)-producing Gram-negatives and methicillin-resistant Staphylococcus aureus (MRSA) accounted for 49 and 41.67%, respectively; 62.2% of the isolates were biofilm formers and were correlated statistically with MDR, ESBL producers, and MRSA (P < 0.005). The extent of biofilm formation and the prevalence of MDR bacteria associated with infected wounds hint at a public health threat that needs immediate attention. Thus, a more balanced and comprehensive wound management approach and antimicrobial stewardship program are essential in the study setting.

Anti-Staphylococcus aureus Activity of Volatile Phytochemicals and Their Combinations with Conventional Antibiotics Against Methicillin-Susceptible S. aureus (MSSA) and Methicillin-Resistant S. aureus (MRSA) Strains.

MSSA and MRSA strains are challenging human pathogens that can develop resistance to antibiotics, highlighting the need for alternative antimicrobial agents. Plant metabolites, particularly volatile phytochemicals, may offer promising antimicrobial properties. The aim was to evaluate the antimicrobial and antibiofilm efficacy of various commercial volatile phytochemicals from the terpene and terpenoid groups against reference MSSA and MRSA strains, focusing on synergistic effects in both binary combinations and combinations with antibiotics. The microdilution method was used to determine the minimum inhibitory concentrations (MICs) for antibiotics and phytochemicals. The checkerboard method assessed synergistic interactions between phytochemicals and between phytochemicals and antibiotics, while the time-kill method was used to confirm these results. Biofilm quantification was performed using the microtiter plate method to evaluate the effects of phytochemicals, antibiotics, and their binary combinations on the eradication of 48-h-old biofilms. Carvacrol and thymol demonstrated the strongest anti-staphylococcal activity, while other terpene compounds showed weaker effects. In binary combinations, carvacrol and thymol exhibited synergy against one MSSA strain (FICI = 0.50) and with tetracycline and chloramphenicol (FICI = 0.28-0.50). Synergy was also noted with streptomycin sulfate against one MRSA strain (FICI = 0.31-0.50) and with other antibiotics, including gentamicin (FICI = 0.25-0.50) and oxacillin (FICI = 0.44). Additionally, effective combinations achieved over 50% biofilm removal at both minimum inhibitory and sub-inhibitory concentrations. Results showed that synergy varies based on strain sensitivity to chemical agents, highlighting their potential for personalized therapy. Despite the difficulty in removing preformed biofilms, the findings highlight the importance of combined treatments to enhance antibiotic effectiveness.

Antibiotic Resistance, Biofilm Formation, and Identification of FimH and FimA Adhesion Genes in Uropathogenic Escherichia Coli (UPEC) Isolated from Patients in Baghdad Province

Urinary tract infections (UTIs) are one of the most common infectious disorders worldwide. The most frequent cause of UTIs is uropathogenic Escherichia coli (UPEC). The current study is designed to assess the resistance of antibiotics, the formation of biofilm, and the detection of adhesion genes in E. coli isolated from patients with UTIs. A hundred and fifty samples were collected from patients with confirmed UTIs in Baghdad province during the period of October 2022 to February 2023. Isolation and identification of E. coli were performed using cultural characteristics, gram staining, and biochemical examination, and the results indicated that 52 (35%) isolates were identified as E. coli. Eleven antibiotic discs were utilized to estimate the sensitivity of E. coli isolates. 98% of isolates were resistant to ampicillin, followed by 75.5% of isolates resistant to both trimethoprim/sulfamethoxazole and fosfomycin, followed by 73.5, 71.4, 67.3, 53.1, and 6.1% of isolates resistant to imipenem, ceftazidime, nitrofurantoin, cefepime, and amikacin. All isolates were sensitive to piperacillin-tazobactam, ciprofloxacin, and aztreonam at 100%, 100%, and 98%, respectively. The microtiter plate method was utilized to estimate the ability of E. coli to form biofilm. The majority of isolates (69.2%; n = 36) were moderate biofilm producers, while 19.2% (n = 10) and 11.5% (n = 6) of isolates were strong and weak biofilm producers, respectively. Polymerase Chain Reactions (PCR) were applied to determine the gene expression level of adhesion genes (fimH and fimA genes). Forty isolates harbored both fimH and fimA genes.

Virulence genes and antibiotic susceptibility patterns of Escherichia coli isolated from nosocomial urinary tract infections in the northwest of Iran during 2022-2023: A cross-sectional study.

Urinary tract infections (UTIs) are prevalent among hospitalized patients, constituting the most frequent health-care infections. Uropathogenic Escherichia coli (UPEC) is leading causative agent of UTIs. The present study was aimed to examine the susceptibility of UPEC isolates obtained from nosocomial cases to antibiotics, as well as their biofilm formation capability and frequency of virulence genes. A total of 100 UPEC isolates were collected from nosocomial UTIs at Imam Reza Hospitals in Tabriz, Iran, spanning from April 2022 to January 2023. The antimicrobial susceptibility patterns were evaluated using the disk diffusion method, along with the detection of broad-spectrum β-lactam enzymes (ESBLs) and carbapenemases. The ability of isolates to form biofilms was assessed using the microtiter-plate method, while the PCR method was employed to identify the presence of virulence genes. The highest resistance was observed toward piperacillin (82%), followed by aztreonam and ciprofloxacin (81%), while the lowest resistance was found against piperacillin/tazobactam (12%) and meropenem (9%). ESBLs were detected in 62% of the isolates. The microtiter-plate results revealed strong, moderate, and weak biofilm formation abilities in 32%, 33%, and 24% of the isolates, respectively. The most prevalent virulence gene was fimA (74%) followed by hlyF (68%), papA (44%), papC (32%), iroN (26%), and cnf (20%). The elevated levels of resistance to multiple antimicrobial agents, coupled with the co-presence of virulence genes and biofilm formation abilities, contribute to the persistence of UPEC-related infections, particularly in hospitalized patients. These findings underscore the necessity of implementing an effective program to control nosocomial UTIs caused by UPEC in the healthcare centers.

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.

Exploring the Antibacterial and Antibiofilm Efficacy of Psammogeton biternatum Edgew and Identification of a Novel Quinoline Alkaloid using X-ray Crystallography.

The prevalence of resistance to harmful human pathogens is steadily rising, emphasizing the urgent need to identify novel antimicrobial compounds. For this purpose, plants stand out as a significant source of bioactives worthy of exploration. Among these, alkaloids, a vast and structurally diverse category of plant secondary metabolites, have emerged as a foundation for crucial antibacterial medications such as metronidazole and the quinolones. In the current work, the crude methanol leaf extract of Psammogeton biternatum Edgew collected from District Bannu, Pakistan, was subjected to TLC (indirect) bioautography and X-ray crystallography for the isolation of potential antibacterial agents. From the crude extract, a novel quinoline alkaloid called quinoline dione ((3R,3aS,5aR)-3,5a,9-trimethyl-3a,4,5,5a-tetrahydro-2H-isoxazolo[2,3-a] quinoline-2,8(3H)-dione (C14H17NO3)) was isolated. The crystal information (M = 247.296 g/mol) is as follows: orthorhombic, P212121, a = 7.7339(14) Å, b = 10.7254(19) Å, c = 15.730(2) Å, V = 1304.8(4) Å3, Z = 4, T = 296 K, μ(Mo Kα) = 0.088 mm-1, ρ calc = 1.259 g/cm3, 13928 reflections measured (5.86° ≤ 2Θ ≤ 51.98°), 2478 unique (R int = 0.1613, R σ = 0.1335). The final R 1 was 0.1098 (I ≥ 2u(I)), and wR 2 was 0.2183. The antibacterial activity for both crude extract of leaves and quinoline dione was determined by a well diffusion method. The quinoline dione alkaloid demonstrated excellent inhibition zones against methicillin-resistant Staphylococcus aureus (18 mm), Bacillus subtills (17 mm), Escherichia coli (20 mm), and Pseudomonas aeruginosa (23 mm) compared to the crude extract. The antibiofilm potential was recorded against Pseudomonas aeruginosa by the 96-well microtiter plate method. A dose-dependent biofilm inhibition response was recorded, which increased with the increase in concentration. Moreover, quinoline dione showed a greater antibiofilm effect as compared to the crude extract, which may be linked to the presence of a particular active functional group positioned on the compound isolated in its pure form. Through in silico studies, i.e., molecular docking, quinoline dione shows strong binding energies with the LasR transcriptional regulator (6MVN) at -9.3 and LasR transcriptional activator (3IX4) at -9.2 kcal/mol, as well as moderate affinities with other targets such as AHL synthase LasI (PDB ID 1RO5) and OprM channel (PDB ID 3D5K), indicating its potential as a quorum sensing inhibitor. Thus, the antibacterial and antibiofilm potential of quinoline dione was confirmed.

Synergistic effect of graphene oxide and silica nanocomposites towards multifunctional biomedical applications

The development of advanced nanomaterials (NMs) has recently gained significant attention because of their potential in addressing various biomedical and healthcare challenges. This study focuses on the synthesis of silica (SiO2) nanoparticles (NPs) and their nanocomposite with graphene oxide (GO-SiO2), using a multifunctional approach to enhance antibacterial, antibiofilm, antioxidant, and antidiabetic properties. The synthesis of the GO-SiO2 involves the controlled growth of SiO2 NPs on the surface of the graphene oxide (GO) sheets. The synthesized NPs were characterized by UV–Visible spectroscopy, Fourier-Transform Infrared (FTIR) Spectroscopy, X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), and Energy Dispersive X-ray (EDX) Spectroscopy. The crystallite sizes of SiO2 and GO-SiO2 were found to be 23.07 nm and 32.07 nm, respectively, by XRD analysis and 27.03 nm and 43.11 nm, respectively, by SEM. The Band gap of SiO2 was decreased from 3.76eV to 3.45eV in GO-SiO2. Disc diffusion method was used to perform antibacterial activity whereas ampicillin and moxifloxacin were taken as reference drugs. Biofilm inhibitions and minimum inhibitory concentrations were measured by microtiter plate method. The composite has shown higher antimicrobial activities against S. aureus, S. aureus MDR, K. pneumoniae, P. aeruginosa, P. aeruginosa MDR, E. coli, and P. vulgaris as compared to SiO2 NPs and GO. When IC50 values were compared with those of control (acarbose), GO-SiO2 and SiO2 demonstrated the highest and lowest antidiabetic activities, respectively. A 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging process was used to evaluate the antioxidant potential of NMs using ascorbic acid as a reference. GO-SiO2 displayed the negligible IC50 value compared to both SiO2 and GO. The nanocomposite (GO-SiO2) has shown higher antibacterial, antibiofilm, antioxidant, and antidiabetic potential as compared to those of GO and SiO2NPs.

Emergence of lineage ST150 and linezolid resistance in Enterococcus faecalis: a molecular epidemiology study of UTIs in Tehran, Iran.

Urinary tract infections (UTIs) represent one of the most prevalent bacterial infections, with Enterococcus species now recognized as the second leading cause of these infections. This study focused on symptomatic UTI cases to investigate the risk factors associated with Enterococcus faecalis clinical isolates in patients from Tehran, Iran. Urine samples were collected from patients presenting with symptomatic UTIs. The identification of E. faecalis isolates was performed using standard microbiological techniques, with confirmation via polymerase chain reaction (PCR). Antibiotic susceptibility testing was conducted using the Kirby-Bauer disc diffusion method. The presence of virulence genes was determined through PCR, and biofilm formation was assessed using the microtiter plate method. Additionally, multi-locus sequence typing (MLST) was utilized to genotype linezolid-resistant isolates. Out of 300 UTI cases, E. faecalis was identified as the causative agent in 160 instances. Notably, a high proportion of these isolates exhibited resistance to tetracycline (83.8%) and minocycline (82.5%). Linezolid resistance was observed in 1.3% (n = 2) of the isolates. Conversely, the highest susceptibility rates were observed for vancomycin, penicillin G, ampicillin, and nitrofurantoin, each demonstrating a 98.8% susceptibility rate. Biofilm formation was detected in 25% of the E. faecalis isolates. A significant majority (93.8%) of the isolates harbored the efbA and ace genes, with varying frequencies of esp (72.5%), asa1 (61.2%), cylA (52.5%), and gelE (88.8%) genes. MLST analysis demonstrated that both linezolid-resistant isolates, characterized by strong biofilm formation and the presence of virulence genes, were assigned to the ST150 lineage, which has not been previously documented in clinical settings. The emergence of the ST150 clonal lineage, underscores its clinical significance, particularly in relation to linezolid resistance in E. faecalis. This study adds to the growing body of evidence linking specific clonal lineages with antibiotic resistance, highlighting the critical need for ongoing surveillance and molecular characterization of resistant pathogens.

Antibacterial and anti-biofilm efficacy of selenium nanoparticles against Pseudomonas aeruginosa: Characterization and in vitro analysis

Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative opportunistic pathogen, poses significant treatment challenges due to its antibiotic resistance and biofilm formation. This study investigates the anti-bacterial and anti-biofilm activities of chemically synthesized selenium nanoparticles (SeNPs) against P. aeruginosa. SeNPs were synthesized using ascorbic acid as a reducing agent and characterized. Biofilm formation was quantified using a modified microtiter plate method, and the anti-biofilm efficacy of SeNPs was evaluated using confocal microscopy and SEM. The P. aeruginosa isolates exhibited high resistance to piperacillin-tazobactam (60 %) and ceftazidime (59 %). SeNPs demonstrated a round shape with a diameter of 15–18 nm. UV–Vis spectra showed a peak at 275 nm, and XRD analysis revealed crystalline peaks corresponding to selenium. The FTIR spectra confirmed the presence of various functional groups. SeNPs significantly reduced biofilm formation in a dose-dependent manner, with MIC50 and MIC90 values of 60 μg/mL and 80 μg/mL, respectively. Confocal microscopy and SEM analysis showed a notable decrease in biofilm thickness and bacterial adherence post-SeNPs treatment. These findings suggest that SeNPs could be a promising alternative or adjunctive treatment option for combating antibiotic-resistant P. aeruginosa infections. Further research is warranted to explore the clinical applications of SeNPs in treating biofilm-associated infections.

Evaluation of Anti-biofilm Activity of Silica nanoparticles against dental Streptococcus mutans: In-Vitro Study

Background: Dental caries is a prevalent chronic infection caused by cariogenic bacteria that cling to teeth, mainly Streptococcus mutans. These bacteria break down sugars into acid over time, demineralizing the tooth structure. Biofilm as an early colonizer and is the most important bacterium in the formation of dental caries . Materials and Methods: The present study concentrates on isolation and identification of the S.mutans pathogen from patients suffering from dental caries. On specific media, the isolates were cultivated. From human teeth, one hundred dental caries samples are gathered. Using MS-agar (Mitis Salivarius agar), only fifty samples are regarded as positive bacterial isolates to identify the S.mutans isolates, their morphological, cultural, biochemical, and VITEK features were examined. . The microtiter plate method was used to detect the development of biofilms by comparing the characteristics of the isolates. In this work, the median values of optical density (OD) at 630 nm were utilised to employ ELISA to distinguish between S. mutans isolates that developed biofilms and those that did not. To confirm the type of bacteria using molecular identification. Objectives: The purpose of this work is to determine the subminimum and minimum inhibitory concentrations of silica nanoparticles that function as anti-biofilm agents, as they have an impact on bacterial growth and details of silica nanoparticle characterization. Results: In this study considered to be S.mutans are twenty isolates the morphological, cultural, biochemical, and VITEK properties of the bacterial isolates were used to identify them . in this study molecular identification to more confirm of S.mutans the result of PCR showed that 20/20(100%) isolates were PCR positive for 16r RNA gene . in this investigation different isolates were evaluated for their capacity to create biofilm; nine (45%) were judged to be strong producers, seven (35%), to be moderate producers, and four (20%) to be weak producers. the sol-gel method was used in this study to synthesis silica nanoparticles, which were then characterised by XRD, FE-SEM, EDX, and TEM. In this study, Si-NPs were tested against isolates of S.mutans by using a resazurin mediated microtiter plate the results have shown that silica nanoparticles has inhibitory action (MIC) against S. mutans with concentration 0,78 mg/ml and sub-MIC with concentration 0.39 mg/ml. .The anti-biofilm effect of silica nanoparticles on isolates of S. mutans in the current investigation revealed that the biofilm mean of control (biofilm development without silica nanoparticles) was 0.583, whereas the findings for anti-biofilm utilizing sub-MIC of NPs, was 0.185 in microtiter plate method when the (p value) less than 0.05 this proves the NPs effected on the biofilm formation by S.murans and inhibition it . Conclusion: Silica nanoparticles' anti-biofilm action on S.mutans isolates demonstrated the importance of biofilm inhibition.