Congo Red Agar Plate Research Articles

Candida species covered from traditional cheeses: Characterization of C. albicans regarding virulence factors, biofilm formation, caseinase activity, antifungal resistance and phylogeny

This study has provided characterization data (carriage of virulence, antifungal resistance, caseinase activity, biofilm-forming ability and genotyping) of Candida albicans isolates and the occurrence of Candida species in traditional cheeses collected from Kayseri, Türkiye. Phenotypic (E-test, Congo red agar and microtiter plate tests) and molecular tests (identification, virulence factors, biofilm-formation, antifungal susceptibility) were carried out. The phylogenetic relatedness of C. albicans isolates was obtained by constructing the PCA dendrogram from the mass spectra data. Of 102 samples, 13 (12.7%) were found to be contaminated with C. albicans, 15 (14.7%), 10 (9.8%) and five (4.9%) were found to be contaminated with C. krusei, C. lusitane and C. paraplosis, respectively. While seven (16.2%) of 43 Candida spp. isolates were obtained from cheese collected from villages, 36 (83.7%) belonged to cheeses collected from traditional retail stores. The carriage rate of C. albicans isolates belonging to virulence factors HSP90 and PLB1 genes was 30.7%. ALST1, ALST3, BCR, ECE, andHWP (virulence and biofilm-associated) genes were harbored by 30.7%, 23%, 38.4%, 53.8%, and 38.4% of the 13 isolates. According to the microplate test, eight (61.5%) of 13 isolates had strong biofilm production. ERG11 and FKS1 (antifungal resistance genes) were found in 46.1% and 23% of 13 isolates, respectively. Due to missense mutations, K128T, E266D and V488I amino acid changes were detected for some isolates regarding azole resistance. As a result of the E-test, of the 13 isolates, one (7.6%) was resistant to flucytosine, four (30.7%) were resistant to caspofungin, and nine (69.2%) were resistant to fluconazole. The PCA analysis clustered the studied isolates into two major clades. C. albicans isolates of traditional cheese collected from villages were grouped in the same cluster. Among the C. albicans isolates from village cheese, there were those obtained from the same dairy milk at different times. Samples from the same sales points produced at different dairy farms were also contaminated with C. albicans. Concerning food safety standards applied from farm to fork, in order to prevent these pathogenic agents from contaminating cheeses, attention to the hygiene conditions of the sale points, conscious personnel, prevention of cross contamination will greatly reduce public health threats in addition to the application of animal health control, milking hygiene, pasteurization parameters in traditional cheese production.

A study of clinical and bacteriological characteristics of Klebsiella pneumoniae isolates in Birjand, South-East Iran: Hypervirulent phenotype, biofilm formation, virulence factors, and antibiotic resistance

This study assessed antimicrobial susceptibility, biofilm formation, hypervirulence traits, and virulence-related genes in K. pneumoniae. 119 isolates underwent antibiotic susceptibility testing using the Kirby-Bauer method, biofilm assessment through Congo red agar and Microtiter plate assays, PCR for various genes, and the String test for hypermucoviscosity. Clinical characteristics and virulence factors of hypervirulent (hvKP) were compared to classic K. pneumoniae (cKP) strains. Among the isolates, 77.3% were multi-drug resistant (MDR), and 83.2% showed biofilm-forming ability, with a higher MDR incidence in biofilm producers. A significant correlation was found between biofilm formation and the presence of the blaCTX-M15 gene. Genes luxS, mrkA, pgaA, and wzm were significantly related to biofilm production. Three K. pneumoniae (2.5%) were identified as hvKP, with higher prevalence of capsular serotypes K1/K2 and virulence-related genes iuc, rmpA, and rmpA2 than cKP. The study underscores the importance of surveillance and stewardship in combating MDR, biofilm-forming, highly virulent K. pneumoniae.

Production and analysis of synthesized bacterial cellulose by Enterococcus faecalis strain AEF using Phoenix dactylifera and Musa acuminata fruit extracts.

Bacterial cellulose (BC) is a highly versatile biopolymer renowned for its exceptional mechanical strength, water retention, and biocompatibility. These properties make it a valuable material for various industrial and biomedical applications. In this study, Enterococcus faecalis synthesized extracellular BC, utilizing Phoenix dactylifera and Musa acuminata fruit extracts as sustainable carbon sources. LC-MS analysis identified glucose as the primary carbohydrate in these extracts, providing a suitable substrate for BC production. Scanning Electron Microscopy (SEM) revealed a network of BC nanofibers on Congo red agar plates. ATR-FTIR spectroscopy confirmed the presence of characteristic cellulose functional groups, further supporting BC synthesis. X-ray diffraction (XRD) analysis indicated a high crystallinity index of 71%, consistent with the cellulose I structure, as evidenced by peaks at 16.22°, 21.46°, 22.52°, and 34.70°. Whole-genome sequencing of E. faecalis identified vital genes involved in BC biosynthesis, including bcsA, bcsB, diguanylate cyclase (DGC), and 6-phosphofructokinase (pfkA). Antibiotic susceptibility tests revealed resistance to cefotaxime, ceftazidime, and ceftriaxone, while susceptibility to imipenem was observed. Quantitative assessment demonstrated that higher concentrations of fruit extracts (5.0-20mg/mL) significantly enhanced BC production. Cytotoxicity testing via the MTT assay confirmed excellent biocompatibility with NIH/3T3 fibroblast cells, showing high cell viability (97-105%). Unlike commonly studied Gram-negative bacteria like Acetobacter xylinum for BC production, this research focuses on Gram-positive Enterococcus faecalis and utilizes Phoenix dactylifera and Musa acuminata fruit extracts as carbon sources. This approach offers a sustainable and promising avenue for BC production.

2,4-Di-Tert-Butylphenol of Streptomyces luridiscabiei MMS-10 Inhibits Biofilm Forming Cariogenic Streptococcus mutans ULSP-2.

Dental caries is a common chronic infectious disease of the oral cavity that affects the overall oral health of the individual. Cariogenic bacteria have long been recognized for their role in developing chronic dental infections. Drug-resistant bacteria represent a global challenge to effective pathogen control in caries. The present study aimed to isolate and identify soil actinomycetes for their antibacterial and anti-biofilm activities against antibiotic-resistant and biofilm-forming cariogenic bacteria. Thirteen caries bacteria isolated from infected tooth samples were evaluated for antibiotic resistance and biofilm formation. The isolate ULSP-2 showed the highest antibiotic resistance score (0.714) and was found to be a strong biofilm producer when tested by congo red agar and microtiter plate assays. The bacterium was identified as Streptococcus mutans based on morphological, biochemical, and molecular characterization. The effect of ethyl acetate extracts from 20 soil actinomycetes on the growth and biofilm formation ability of S. mutans was evaluated. The MMS-10 extract strongly inhibited growth (18.5 ± 0.5mm) and biofilm formation (56.46 ± 0.32%) of S. mutans at 100µg/mL. The isolate MMS-10 was identified at the molecular level as Streptomyces luridiscabiei. Based on FTIR, NMR, and GC-MS analysis, the purified MMS-10 extract was characterized and identified as 2,4-Di-tert-butylphenol. The metabolite's physiological, physicochemical, and pharmacokinetic properties were analyzed using the Swiss ADME web server and found to satisfy the criteria of drug-likenessof a molecule. The study revealed the significance of soil actinomycetes in controlling growth and biofilm formation in cariogenic S. mutans.

Evaluation of Antimicrobial Activity of Selected Plant Extracts against Staphylococcus Strains Isolated from Wound Infections.

Wound infections are a significant medical problem, with Staphylococcus species being one of the most common etiological factors. Treatment is complex due to bacterial antibiotic resistance and the ability to form a biofilm. The aim of this study was to determine the drug susceptibility of the clinical isolates of Staphylococcus species obtained from wound infections, assess their ability to form a biofilm in vitro, evaluate the level of extracellular slime synthesis, and test the antistaphylococcal properties of selected plant extracts against plate-cultured bacteria and activity against mature biofilms. A total of 20 Staphylococcus strains were evaluated. The antibiotic susceptibility and sensitivity of the strains to the tested extracts were determined using the disc diffusion method. The production of extracellular bacterial slime was assessed using Congo Red agar plates. The biofilm formation and the effect of plant extracts on the biofilm layer were examined using the MTT method on polystyrene microtiter plates. Diverse drug susceptibility profiles, slime production ability, and in vitro biofilm formation were observed among the tested strains. It was found that aqueous extracts from the Serpylli herba, Arctii lappae folium, Taraxaci folium, and Galii aparini herba showed antibacterial activity against some of the tested strains. In contrast, their antibiofilm activity was not confirmed. Among all tested preparations, the most promising antimicrobial extracts in both planktonic cultures and biofilm were Thymus serpyllum herb and Taraxacum officinale leaf extracts. These results allowed us to conclude their potential application in medicine to support the treatment of challenging wound conditions.

Assessment of prevalence, antibiotic resistance, and virulence profiles of biofilm-forming Enterococcus faecalis isolated from raw seafood in Bangladesh

Enterococcus faecalis are often resistant to different classes of antibiotics, harbor virulence determinants, and produce biofilm. The presence of E. faecalis in raw seafood exhibits serious public health significance. This study aimed to identify antibiotic resistance patterns and virulence factors in biofilm-forming E. faecalis strains extracted from seafood in Bangladesh. A total of 150 samples of raw seafood, comprising 50 shrimps, 25 crabs, and 75 fish, were collected and subjected to culturing, biochemical, and PCR assays to detect E. faecalis. The biofilm-forming abilities of the isolates were determined by Congo Red agar (CRA) plate and Crystal Violet Micro-titer Plate (CVMP) tests. Antibiotic resistance profiles were evaluated using the disk diffusion method. Virulence genes of the isolates were detected by PCR assay. The occurrence of E. faecalis was 29.3 % (44/150), which was higher in crabs and fish (36 %) than in shrimps (16 %). In CRA and CVMP tests, biofilm-forming abilities were observed in 88.64 % of the isolates, whereas 11 (25 %) and 28 (63.6 %) were strong- and intermediate-biofilm formers, respectively. All the isolates contained at least two virulence genes, including pil and ace (97.7 %), sprE (95.5 %), gelE (90.9 %), fsrB (79.6 %), agg (70.5 %), fsrA (68.2 %), and fsrC (61.4 %). All the isolates were phenotypically resistant to penicillin, followed by ampicillin and rifampicin (86.4 %), erythromycin (13.7 %), and tetracycline, vancomycin, norfloxacin, and linezolid (2.3 %). Resistant gene blaTEM was found in 61.4 % of the isolates. Moreover, the study found that E. faecalis strains with strong biofilm-forming capabilities had significantly higher levels of virulence genes and antibiotic resistance (p < 0.05) compared to those with intermediate and/or no biofilm-forming abilities. To the best of our knowledge, this research represents the first instance in Bangladesh of assessing antibiotic resistance and identifying virulence genes in biofilm-forming E. faecalis strains isolated from seafood samples. Our study revealed that seafood is a carrier of antibiotic-resistant, virulent, and biofilm-forming E. faecalis, demonstrating a potential public health threat.

Molecular and phenotypic characterization of biofilm formation and antimicrobial resistance patterns of uropathogenic staphylococcus haemolyticus isolates in Casablanca, Morocco

This study aimed to establish the correlation between antibiotic resistance and biofilm formation by Staphylococcus haemolyticus and to examine the impact of sub-inhibitory concentrations of antibiotics (sub-MICs) on biofilm formation.Antibiotic susceptibility testing was conducted using the disk diffusion method, and biofilm formation was determined using Congo red agar and microtiter plate methods. Antibiotic resistance and biofilm-associated genes were detected using polymerase chain reaction.The majority of the twenty-one S. haemolyticus isolates were multidrug-resistant, methicillin-resistant (MRSH) and biofilm producers, including 43 % of moderate biofilm producers. A significant correlation was observed between MRSH and MSSH isolates in terms of biofilm production. Vancomycin, gentamicin, and ciprofloxacin at their sub-MICs tended to promote biofilm formation. The eno gene was present in 76.2 % of strains, followed by aap, and atlE.This study revealed a strong correlation between the biofilm-forming ability and antibiotic resistance in S. haemolyticus, which underlines a crucial public health issue.

Determine Biofilm Genes in Pseudomonas Aeruginosa Isolated from Clinical and Environmental Samples

Objective: Isolates of Pseudomonas aeruginosa are an extremely adaptable bacterium that causes opportunistic diseases because of its varied metabolic pathways, genes, virulence factors, and considerable antibiotic resistance. Methods: A total of 293 samples were collected from different places: 193 samples (% 65.87) of human samples and 100 samples (% 34.13) of wastewater samples in the period between 3rd September to 15th November 2023). Bacterial isolates were identified according to microscopic, cultural, and genetic characteristics. Antibiotic susceptibility of bacterial isolates was determined against twelve of the selected antibiotics. The biofilm production was done by using phenotypic ways (Congo red agar and Microtiter plate methods) as well as genotypic ways by detection of biofilm genes (algD, pelf, and pslD genes). Results: Hundred-forty eight bacterial isolates were obtained, and sixty of these isolates were identified as Pseudomonas spp. (40.9%), twenty-six isolates of E. coli (17.9%), seventeen isolates of K. pneumoniae (11.3%), and forty-five isolates were beyond to other types of bacteria (30.1%), and out of sixty isolates of Pseudomonas spp., forty-two were identified as P. aeruginosa isolates. P. aeruginosa isolates revealed various resistance levels to antimicrobial agents gradually, ranging from 83.87% to Trimethoprim-sulfamethoxazole (SXT) to 3.22% to Aztreonam (AZT). Biofilm production by using the Congo red method showed that 27 isolates (64.28%) were positive results, while in the microtiter method, all forty-two isolates were positive (100%), the genetic detection showed that the AlgD gene was recognized in thirty-one isolates (73.8%), followed by Pelf and PslD genes in four isolates each (9.5%). Conclusion: The isolation percentage showed a high occurrence of multi-drug resistance biofilm forming Pseudomonas spp. isolates which could be a critical indicator. Methods of biofilm detection showed that the microtiter plate method has accuracy more than the Congo red method; as well AlgD gene was prevalent compared with both other genes Pelf, and PslD.

High Biofilm-Forming Multidrug-Resistant Salmonella Infantis Strains from the Poultry Production Chain.

The ability of Salmonella species to adhere to surfaces and form biofilms, leading to persistent environmental reservoirs, might represent a direct link between environmental contamination and food processing contamination. The purpose of this study was to investigate the biofilm-forming ability of 80 multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL) producing Salmonella enterica serovar Infantis strains isolated from the broiler food chain production through whole genome sequencing (WGS), PCR, and morphotype association assays. Biofilm formation was quantified by testing the strains at two different temperatures, using 96-well polystyrene plates. The rough and dry colony (rdar) morphotype was assessed visually on Congo red agar (CRA) plates. Based on our results, all tested S. Infantis strains produced biofilm at 22 °C with an rdar morphotype, while at 37 °C, all the isolates tested negative, except one positive. Most isolates (58.75%) exhibited strong biofilm production, while 36.25% showed moderate production. Only 5 out of 80 (6.25%) were weak biofilm producers. WGS analysis showed the presence of the fim cluster (fimADF) and the csg cluster (csgBAC and csgDEFG), also described in S. Typhimurium, which are responsible for fimbriae production. PCR demonstrated the presence of csgD, csgB, and fimA in all 80 S. Infantis strains. To our knowledge, this is the first study comparing the effects of two different temperatures on the biofilm formation capacity of ESBL producing S. Infantis from the broiler production chain. This study highlights that the initial biofilm components, such as curli and cellulose, are specifically expressed at lower temperatures. It is important to emphasize that within the broiler farm, the environmental temperature ranges between 18-22 °C, which is the optimum temperature for in vitro biofilm formation by Salmonella spp. This temperature range facilitates the expression of biofilm-associated genes, contributing to the persistence of S. Infantis in the environment. This complicates biosecurity measures and makes disinfection protocols on the farm and in the production chain more difficult, posing serious public health concerns.

Biofilm generation and antibiotic resistant profile of extensive and multidrug resistant Pseudomonas aeruginosa from burn patients in Ahvaz: A cross-sectional study.

Multidrug and extensive drug-resistant Pseudomonas aeruginosa was extracted from burn patients referring to burn centers in southwest Iran so that biofilm generation and antibiotic resistance could be investigated. A specific primer was used to confirm all our considered 110 P. aeruginosa culture-positive reports on 345 burn patients. The resistance of P. aeruginosa to seven antibiotics and Colistin with minimum inhibitory concentration (MIC) was assessed. Biofilm formation was assessed by the phenotypic study of specimens under Congo red agar and microtiter plate assays. One hundred and 10 clinical P. aeruginosa isolates taken from burn wound infections were validated. Among P. aeruginosa isolates, Piperacillin, Ceftazidime, Maeropenem, Gentamycin, and Gatifloacin had the highest resistance to antibiotics, while Ticarcillin-Clavulanic acid and Ceftolozane-Tazobactam showed the least resistance. MICs were then evaluated via the E test. Seven isolates were resistant to colistin. Colistin reference MICs for multidrug-resistant P. aeruginosa prevalence was 38%, while it was 22% for extensively drug-resistant (XDR) P. aeruginosa. One P. aeruginosa was pandrug-resistant (PDR). Under Congo red agar test, 66 isolates (67%) formed biofilms and black colonies, whereas 44 isolates (50%) had red colonies. In MTP, 76% formed biofilm. 40%, 32%, 21% of the isolates were strong, moderate, and weak biofilm formers, respectively, while 43% did not form biofilms. The P. aeruginosa resistance to antimicrobial agents has largely challenged the control of the infection. Accordingly, a higher resistance occurred when the isolates were transferred to the patients. Less than 50% P. aeruginosa samples generated strong biofilms. Consequently, hygienic measurements are essential to inhibit P. aeruginosa transmission to hospitalized patients.

Effect of Sulphur Nanoparticles Biosynthesized from Bacillus Coagulans Extract against S. aureus Isolated from Dermatitis Patients in Iraqi Hospitals

Objective: The goal is to biosynthesize sulphur nanoparticles (Sulphur NPs)from Bacillus coagulans extract and test its bioactivity against S. aureus gram positive bacteri that was isolated from dermatitis patients. Methods: 125 specimens were taken from patients of dermatitis attended two Baghdad hospitals. Among 125 specimens, only 32 of isolates were identified as S. aureus. Five S. aureus isolates biofilm forming(strong) and multi drug resistant(MDR) were selected. Fourteen types of antibiotics from different classes were used and the results showed different patterns of resistance. Congo red agar (CRA) and microtiter plate method (MPT) were used to estimate the ability of MDR isolates of S. aureus to create biofilms. A series of characterization techniques, including UV–vis, FTIR, FE-SEM, EDS, XRD and TEM analysis, have been carried out to characterize the biosynthesized sulfur NPs. The antimicrobial, antibiofilm and antioxidant activities of sulfur NPs were estimated. Results: Sulfur NPs biosynthesized exhibited antimicrobial activity against examined bacteria with inhibition zone ranged between 12 to 20, 18 to 23 and 22 to 26mm at 100, 200 and 300µg/ml, respectively. Only five strong-biofilm producers and MDR of S. aureus(A5, A8, A9, A11 and A29) were used to assess the ability of sulfur NPs as anti-biofilm agent. No significant differences (p&gt;0.05) in inhibition rate of all strain isolates (A5, A8, A9, A11 and A29) for all concentrations of Sulfur NPs with p=0.886. Conclusions: The antioxidant activity of sulfur NPs were decreased into 81.6, 80.3, 75.2 and 68.9% with using concentrations of 400, 300, 200 and 100ug/ml, respectively, also, nanoparticles had the potential to scavenge DPPH free radicals.

Phenotypic and Genotypic Study of Staphylococcus Haemolyticus and Staphylococcus Warneri Clinically Isolated from the Bloodstream

Background: The Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci (CoNS) as part of human skin’s natural flora. In recent years, CoNS has increased its visibility as opportunistic pathogens acquired in hospital patients. This study aims to isolate S. haemolyticus and S. warneri strains from patients with bloodstream infections and to analyze the antimicrobial profile of these bacteria by molecular detection of the mecA gene. Materials and Methods: Four hundred and twenty-three clinical blood samples were collected from patients admitted to general three hospitals in Hilla city, Iraq, during the period from September 2021 to July 2022. Blood samples were inoculated immediately into blood bottles containing 70 ml of Brain-Heart Infusion Broth. All isolates were identified by morphology, biochemical tests, and VITEK 2 automated system (bioMerieux, France). The Congo red agar plate test was used to detect biofilm production. The mecA gene was detected by conventional polymerase chain reaction. Results: S. haem olyticus (nine isolates) and S. warneri (six isolates) were detected out of 423 clinical blood samples. Molecular detection of the mecA gene was detected in all 15 isolates. As well 11 (73.3%) isolates were β-lactam resistant and biofilm production. The results of oxacillin-resistance minimum inhibitory concentration (MIC) (≥4 mg/ml) showed that 11 (73.3%) of the 15 isolates were oxacillin and methicillin-resistant CoNS (MR-CoNS). Susceptibility to various isolates (MICs µg/ml) using VITEK 2 system showed erythromycin (≥8 µg/ml), rifampin (≥4 µg/ml), clindamycin (≥4 µg/ml), and trimethoprim-sulfamethoxazole (≥4 µg/ml) resistance were (80%), (13.3%), (20%), and (53.3%), respectively. All isolates were (100%) sensitive to linezolid (≥8 µg/ml) and teicoplanin (≥32 µg/ml). Conclusions: The S. haemolyticus and S. warneri are serious infections associated with bacteremia and septicemia infections. Linezolid, teicoplanin, and rifampin are the drug of choice for the treatment of infections caused by MR-CoNS isolates. A high rate of the mecA gene and biofilm production is present in these bacteria.

In vitro ANALYSIS OF BIOFILM FORMATION AND ANTIBIOTIC RESISTANCE OF UROPATHOGENIC Escherichia coli

Urinary tract infections are an exceedingly common worldwide problem, caused mostly by Gram-negative bacteria, especially Escherichia coli. Microbial biofilms are considered a serious public health problem. The potential of uropathogenic E. coli (UPEC) to produce biofilm was explored in the present study. The Congo red agar, tube- and tissue culture plate methods were used to evaluate the formation of biofilm by E. coli isolates. Of the 155 isolates, 101, 106 and 90 isolates were positive for these three methods, respectively. Subsequently, the sensitivity of E. coli isolates to antimicrobial agents (amikacin, cefepime, cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, ciprofloxacin, nitrofurantoin, gentamycin, nalidixic acid, ofloxacin and pefloxacin) was tested. There was no difference in the rate of biofilm detection between Congo red agar method and tube method. The antibiotic sensitivity test revealed that the biofilm producing isolates were multi-drug resistant. The study emphasized the necessity for developing alternative therapeutic approaches to overcome multi drug resistance arising from biofilm formation of UPEC.

Grewia tenax bark extract mediated silver nanoparticles as an antibacterial, antibiofilm and antifungal agent

Grewia tenax plant possesses many medicinal qualities that can be harnessed for bio-applications via nanotechnology in a cost-effective and environment-friendly manner. The synthesised silver nanoparticles (GTB-AgNPs) from Grewia tenax bark extract were first detected by observing the change in colour from yellowish brown to dark brown and confirmed by UV–vis spectroscopy. UV–vis spectral analysis recorded the surface plasmon resonance at 420 nm. In the current research, several process parameters (time, temperature, concentration of AgNO3 and the ratio of extract to AgNO3) were adjusted by using one variable at a time (OVAT) approach. Spectroscopy accompanied by dynamic light scattering (DLS), x-ray diffraction (XRD) and scanning electron microscopy (SEM) with energy dispersive x-ray (EDX) determines the size, nature, morphological variations and chemical constituents of GTB-AgNPs. The size range was found within 35−45 nm and the image clearly showed that the GTB-AgNPs were spherical in shape and well dispersed in nature. Zeta potential and Fourier-transform infrared spectroscopy (FTIR) were done to analyse GTB-AgNPs stability, dispersion and the biomolecules in aqueous bark extract liable for the bioreduction of silver particles. These GTB-AgNPs showed excellent antibacterial action against Gram-positive (S. aureus and B. subtilis) and Gram-negative bacteria (P. aeruginosa and E. coli) which was observed by disc diffusion assay. Congo red agar plate (CRA) assay effectively unveiled the antibiofilm proficiency of the particles. These studies displayed a reduction in bacterial sustainability and the formation of exopolysaccharides. Disc diffusion assay confirmed the antifungal competency against A. niger and C. albicans. SEM was performed to elucidate the intracellular breakage and morphological transformations in cells of Gram-positive bacteria and fungi after treatment with GTB-AgNPs. The current study, therefore, enlightened applications of GTB-AgNPs as an efficient antimicrobial agent and applicable substitute in the pharmaceutical area.

Epithelial Cell Infection Analyses with Shigella.

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

Catheter-associated urinary tract infection and biofilms: Dreaded duo in health settings

Abstract Background: Urinary tract infections (UTIs) alone account for 30% of hospital infections, 80% of these are catheter-associated UTIs (CAUTI). Microbial film in indwelling medical devices worsens the existing medical condition and leads to multidrug resistance (MDR) and prolonged hospital stay. This cross-sectional study was done to find out the organisms causing CAUTI, their sensitivity pattern, the proportion of biofilm formation, and the sensitivity, specificity of different procedures in the demonstration of biofilm. Materials and Methods: Urine sample was collected from 307 patients with indwelling catheters. Direct Gram stain was performed from well-mixed uncentrifuged urine. Specimens were processed, organisms were isolated by standard microbiological procedures, and antimicrobial sensitivity testing was performed. Various methods such as Congo red agar, tube adherence, and microtiter plate methods were used to identify biofilm formation for all the isolates. Results: Out of 307 catheterized urine samples, a direct Gram stain was positive in 137 (44.62%) samples and culture positive in 148 (48.2%) samples. Out of 134 bacterial isolates, biofilm was observed by Congo red agar in 36 (27%), tube adherence in 59 (44%), and microtiter plate in 80 (60%) isolates. Escherichia coli (50.74%) was the predominant isolate followed by Klebsiella spp (26.11%). MDR was observed in 53 (66%) biofilm-forming isolates. Conclusions: Catheterized patients are prone to develop a microbial biofilm that is multidrug resistant and burdensome. Of the 80 biofilm-producing isolates, MDR was observed in 53 (66%). The effective ways to prevent CAUTI are strict adherence to indications for catheterization and, a bundle care approach with a preinsertion checklist.

Characterization of biofilm formation in uropathogenic Staphylococcus aureus and their association with antibiotic resistance

IntroductionThe increase in antibiotic resistance and biofilm formation in Staphylococcus aureus is a serious public health problem, complicating the treatment of infections caused by these bacteria, including urinary tract infections (UTIs). This research aimed to assess the correlation between antibiotic resistance and the capacity for biofilm formation in S. aureus strains isolated from patients with UTIs. MethodsThe antibiotic susceptibility testing was conducted using disc diffusion and broth micro-dilution method. The blaZ and mecA genes were investigated in all penicillin and cefoxitin-resistant S. aureus strains, using PCR. Biofilm formation was investigated using the Congo red agar and microtitre plate methods. All S. aureus strains were screened for genes encoding adhesion and biofilm formation (spa, fnb AB, clfA, bap) and intercellular adhesin (icaADB and icaR). ResultsAmong 20 strains of S. aureus, 55% were found to be multidrug-resistant (MDR) and methicillin-resistant (MRSA). All strains were biofilm producers, including 55% moderate biofilm producers and 35% strong biofilm producers. A significant correlation (p-value <0.01) was observed between MDR and non-MDR strains in terms of biofilm production. All strains were sensitive to linezolid, and 20% exhibited the constitutive clindamycin resistance phenotype. The spa, fnbB, and clfA genes were present in all strains followed by the bap (95%) and fnbA (90%). The icaA (90%) and icaB (60%) genes were most detected. The coexistence of icaA, icaADB, and icaAD genes was significantly associated with MRSA strains. In contrast, the icaR gene was significantly detectable in the methicillin-susceptible group of strains. ConclusionThis study uncovered a significant association between the formation of biofilms by uropathogenic S. aureus and antibiotic resistance, highlighting a noteworthy concern for public health.

The Antimicrobial of Selenium Nanoparticles and Combination with Cinnamon Oil Against Salmonella Typhi Which Cause Diarrhea in Human

Many bacterial species can cause diseases in humans, such as the bacteria that cause diarrhea as a result of having genetic characteristics that can form pathogenic factors, which help the bacterium to produce harmful effects in the host's body. From (60) fecal samples of diarrheal cases of human, Salmonella spp.42(70%) where Salmonella typhi consist29(48.33%) isolates and Salmonella paratyphi consist13(21.67%) isolates, E. coli 10(16,66%), Enterobacter 4(6,66%) and Shigella 4(6,66%). On Congo red agar (CRA) plates, among (29) S. typhi bacterial isolates from human, all the isolates (100%) were showed biofilm formation through formation of brown colonies with a dry crystalline quality. Antibiotic susceptibility profile of Salmonella typhi. isolated from fecal sample of human were determined by using Vitek2 system, the results revealed that Salmonella typhi were100% resistant to Ticarcillin and 89.7% to Aztreonam while the rest of the antibiotics were less resistant. Selenium nanoparticles (Se-NPs) are green synthesized using cinnamon extract. The antibacterial activity of the cinnamic ethanolic extract and synthesized SeNPs was investigated against isolates of human source Salmonella typhi by using the agar well diffusion method and tested by using different concentrations (25,50,100 μg/mL).According to the data, cinnamon extract only produced an inhibition of 10.66 mm at the same dose of 100 mg/ml, but Se-NPs produced a good inhibition at that concentration of 14.33 mm.

Process optimization of malachite green degradation by mixed biofilm positive bacteria: Application of Box–Behnken designs, RSM and whole genome sequences analysis

Bacterial biofilm positive strains recovered from engine oil polluted-soil was investigated for its capacity to remove malachite green (MG) in mineral salt solution. Box–Behnken designs combining four factors (glucose, yeast extract, temperature and agitation) with three levels (−1, 0, +1) was used to found the best biotic and abiotic conditions for MG removal. The biodegradation potential of the selected microbial consortium was also confirmed using analytical techniques. Then, whole genome sequencing (WGS) analysis were performed to detect genes related to biofilm formation and chemicals biodegradation. Our results showed that the selected strains (G5, G13 and G15) were able to form biofilm on congo red agar plates developing almost black, very black and almost black colonies respectively. Experimental results using Box-Behnken designs showed that decolorization rate of MG reach 89.35% in these optimized conditions: glucose (2%), yeast extract (1%), temperature (40 °C), and agitation (150 rpm). Molecular investigation revealed that the isolated strains were identified as Cronobacter sakazakii G5, Enterobacter hormaechei G13, and Klebsiella pneumoniae G15 based on the whole genome sequences . Whole genome sequences analysis demonstrated the presence of genes involved in biofilm formation as well as dyes and chemical detoxification. The findings presented here provide useful information for development an efficient microbial consortium, formed by adherent bacteria, able to remove dye from coloured wastewater.

Isolation of Biofilm-Forming Staphylococci from the Bulk-Tank Milk of Small Ruminant Farms in Greece.

The objectives of this study were (i) to describe staphylococcal isolates recovered from bulk-tank raw milk collected from sheep and goat farms during a countrywide study performed in Greece, (ii) to study management factors potentially associated with their presence in bulk-tank milk and (iii) to provide evidence regarding their association with the quality of the milk. In total, 312 staphylococcal isolates, recovered from samples of bulk-tank raw milk from 444 small ruminant farms in Greece, were evaluated in this work. The in vitro formation of biofilm by the isolates was tested by combining the findings of (a) culture appearance on Congo Red agar plates and (b) results of a microplate adhesion test. The most frequently identified species was Staphylococcus aureus (75 isolates); other frequently recovered species were S. simulans (44 isolates), S. equorum (34 isolates) and S. haemolyticus (26 isolates); in total, 23 species were identified. In total, 224 (71.8%) isolates were biofilm-forming and were recovered from the bulk-tank milk samples of 148 sheep flocks (45.5%) and 55 goat herds (46.2%). There was evidence of seasonality in the isolation of staphylococci: during spring, mostly biofilm-forming isolates were recovered, whilst during summer, mostly non-biofilm-forming isolates were recovered. Among farms applying machine-milking, the proportion of farms from which biofilm-forming isolates were recovered was higher where water with temperature < 50 °C or ≥90 °C was used to clean the milking parlour. In the multivariable analyses, for farms applying machine-milking, the temperature of the water emerged as the only significant variable (p = 0.024), whilst in farms applying hand-milking, the only tendency that emerged was for the frequency of collection of milk from the farm tank (p = 0.08). In sheep flocks, recovery of biofilm-forming staphylococci from the bulk-tank milk was associated with higher somatic cell counts and higher total bacterial counts in the milk. The study identified abiotic factors related to the presence and isolation of these bacteria, specifically the temperature of water used for the cleaning of the milking parlour (in farms where machine-milking is applied) and the frequency of milk collection from the farm tank. These factors apply after the production of milk, and they could thus be regulated appropriately in order to reduce bacterial load and improve the quality of milk delivered to dairy plants. In sheep farms, an association was also seen between recovery of biofilm-forming staphylococci and high somatic cell counts in milk.