Microtiter Plate Method Research Articles

Characterization of Clonal Groups of Antibiotic-Resistant Biofilm-Forming Staphylococcus aureus Strains Isolated from Patients with Urinary Tract Infections in Isfahan, Iran

Background: Over the past decades, the role of biofilm-forming Staphylococcus aureus strains in urinary tract infections (UTIs) has garnered significant attention. Objectives: This study aimed to determine the epidemiological characteristics and diversity of S. aureus strains isolated from patients with UTIs in Isfahan, Iran, in 2017, with regard to their antimicrobial resistance, biofilm formation, and phylogenetic profiles. Additionally, the study investigated potential relationships among these factors statistically to develop efficient control and treatment approaches. Methods: All patients with symptomatic UTIs who had positive urine cultures for S. aureus during the study period at the laboratory of a referral hospital in Isfahan were included. All isolates were identified using specific primers for the nucA gene. Their biofilm formation capacity was evaluated using a combination of the microtiter plate and Congo-red agar methods. Antibiotic susceptibility testing was performed using the disk diffusion method. The presence of genes involved in biofilm formation and resistance to cefoxitin, aminoglycosides, and fluoroquinolones was detected using polymerase chain reaction (PCR). Staphylococcal cassette chromosome mec (SCCmec) typing, agr typing, and phene plate (PhP) typing were employed to investigate the diversity of collected strains. Results: Results showed that 19%, 57%, and 24% of confirmed S. aureus strains were strong, intermediate, and non-biofilm formers, respectively. The highest rate of resistance was against nalidixic acid (77%), followed by streptomycin (73%). The icaD and icaA genes had the highest frequency among biofilm-producing strains. gyrA (44%) and grlA (35%) were the most frequent genes among fluoroquinolone-resistant strains, while aph(3′)-IIIa was the most prevalent aminoglycoside-modifying enzyme gene. The majority of bacterial strains harbored SCCmec type III and agr type I. PhP typing of strains revealed the presence of 8 common types (CTs) and 14 single types (STs), with CT2 being the dominant type. Conclusions: The present investigation revealed various biofilm production capacities, antimicrobial resistance profiles, and clonal lineages in S. aureus isolated from patients with UTIs. These findings provide further insights into the epidemiology and pathogenicity of S. aureus strains in Iran, thereby improving the quality of surveillance and therapeutic protocols.

Prevalence of plasmid-mediated quinolone resistance genes and biofilm formation in different species of quinolone-resistant clinical Shigella isolates: a cross-sectional study

BackgroundThe purpose of this study was to look into the presence of plasmid-mediated quinolone resistance (PMQR) genes and biofilm formation in several species of clinical Shigella isolates that were resistant to quinolones.MethodsThe stool samples of 150 patients (younger than 10 years) with diarrhea were collected in this cross-sectional study (November 2020 to December 2021). After cultivation of samples on Hektoen Enteric agar and xylose lysine deoxycholate agar, standard microbiology tests, VITEK 2 system, and polymerase chain reaction (PCR) were utilized to identify Shigella isolates. The broth microdilution method was used to determine antibiotic susceptibility. PMQR genes including qnrA, qnrB, qnrC, qnrD, qnrE, qnrS, qnrVC, qepA, oqxAB, aac(6′)-Ib-cr, and crpP and biofilm formation were investigated in quinolone-resistant isolates by PCR and microtiter plate method, respectively. An enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique was used to determine the clonal relatedness of quinolone-resistant isolates.ResultsA total of 95 Shigella isolates including S. sonnei (53, 55.8%), S. flexneri (39, 41.1%), and S. boydii (3, 3.2%) were identified. The highest resistance rates of the isolates were against ampicillin (92.6%, n = 88/95). Overall, 42 of 95 (44.2%) isolates were simultaneously resistant against two or more quinolones including 26 (61.9%) S. sonnei and 16 (38.1%) S. flexneri. All isolates were multidrug-resistant (resistance to more than 3 antibiotics). The occurrence of PMQR genes was as follows: qnrS (52.4%), qnrA and aac(6′)-Ib-cr (33.3%), and qnrB (19.0%). The prevalence in species was as follows: 61.5% and 37.5% (qnrS), 19.2% and 56.3% (qnrA), 38.5% and 25.0 (aac(6′)-Ib-cr), and 19.2% and 18.8% (qnrB) for S. sonnei and S. flexneri, respectively. The other PMQR genes were not detected. In total, 52.8% (28/53) of quinolone-susceptible and 64.3% (27/42) of quinolone-resistant isolates were biofilm producers. Biofilm formation was not significantly different between quinolone-resistant and quinolone-susceptible isolates (P-value = 0.299). Quinolone-resistant isolates showed a high genetic diversity according to the ERIC-PCR.ConclusionIt seems that qnrS, qnrA, and aac(6′)-Ib-cr play a significant role in the quinolone resistance among Shigella isolates in our region. Also the quinolone-resistant S. flexneri and S. sonnei isolates had a high genetic diversity. Hence, antibiotic therapy needs to be routinely revised based on the surveillance findings.

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.

Agroclimatic zone-wise prevalence of biofilm forming methicillin resistant staphylococcus aureus in West Bengal

The present study envisages the prevalence of biofilm producing, methicillin resistant Staphylococcus aureus, isolated from cattle milk of different agro-climatic zones of an eastern state of India, West Bengal. The milk samples (n=168) were collected from three predominant agro- climatic zones of South Bengal, viz. new alluvial, coastal saline and red laterite. A total of 146 Staphylococcus aureus isolates were recovered, confirmed by biochemical tests and polymerase chain reaction (PCR). Through PCR of mecA gene, it was found that 45 strains were methicillin resistant, encompassing 28.57% new alluvial zone, 30.19% red laterite region and 29.16% coastal saline region. Determination of biofilm formation was done, both phenotypically by microtiter plate method and genotypically by PCR of icaA and icaD genes, specific for biofilm formation in Staphylococcus aureus. Phenotypic study revealed that 14 isolates were strong biofilm producers, 79 were moderate and the 47 were weak biofilm producers. PCR studies also showed that a total of 30 isolates were icaA positive and 25 isolates were icaD positive. Although no significant difference was found in the occurrence of icaA positive isolates, the possession of icaD by the Staphylococcus aureus isolates varied significantly according to different agro-climatic zones.

Interactive effects of osmotic, acid, heat, cold, and freezing stresses on the biofilm formation ability of Salmonella serotypes

AbstractFood‐related stresses such as heating and freezing may influence the biofilm formation ability of bacteria. This study aimed to investigate the main and interactive effects of food‐related stresses on the biofilm formation potential of Salmonella strains isolated from meat. Salmonella enteritidis, Salmonella typhi, and Salmonella typhimurium were subjected to osmotic, acid, heat, cold, and freezing stresses. The colorimetric microtiter plate method was used to measure the biofilm formation ability as a response to the stresses. Among the main effects, freezing time had the most significant effect on the biofilm formation responses of three Salmonella serovars. Freezing reduced the biofilm formation ability of Salmonella typhimurium and Salmonella enteritidis but increased that of Salmonella typhi (p < 0.05). Among the interaction effects, the most significant effect on the biofilm formation response of Salmonella typhi was the interaction between pH and heat, which had a negative effect on the biofilm formation response. In contrast, the interaction between osmotic pressure and cold stresses was the most significant interactive effect on the biofilm formation responses of Salmonella enteritidis and Salmonella typhimurium, which had an increasing effect. This study concluded that the food‐related stresses could change the biofilm formation capacity of Salmonella serotypes, and each serotype might show different biofilm formation abilities in response to different stresses.

Molecular characterization, antibiotic resistant pattern and biofilm forming potentiality of bacterial community associated with Ompok pabda fish farming in southwestern Bangladesh

Ompok pabda is gaining popularity in the aquaculture industry due to its increasing demand; however research on microbial diversity and antibiotic susceptibility remains limited. The present study was designed to identify the bacterial pathogens commonly found in the pabda farming system with their biofilm forming potential and antibiotic susceptibility. Different bacterial strains were isolated from water, sediments and gut, gill of pabda fish and the isolates were identified based on their morphological traits, biochemical and molecular analysis. Antibiotic susceptibilities, antibiotic resistance gene determination and biofilm formation capabilities were evaluated by disc diffusion method, PCR amplification and Microtiter plate (MTP) assay, respectively. The respective isolates of gill and gut of pabda aquaculture and their environments were: Exiguobacterium spp. (25 %), Enterococcus spp. (20 %), Bacillus spp. (10 %), Acinetobacter spp. (10 %), Enterobacter spp. (10 %), Aeromonas spp. (10 %), Lactococcus spp. (5 %), Klebsiella spp. (5 %) and Kurthia spp. (5 %). Antibiotic resistance frequencies were found to be relatively high, especially for trimethoprim (95 %), sulfafurazole (75 %), ampicillin (60 %), amoxicillin-clavulanic acid (55 %), and cephradine (50 %). 30 % isolates were categorized as DR bacteria followed by 30 % isolates were MDR bacteria and 40 % were classified as XDR bacteria. Moreover, 4 antibiotic resistant genes were detected with sul1 (30 %), dfrA1 (10 %), tetC (40 %), and qnrA (5 %) of isolates. Based on the microtiter plate method, 20 %, 25 %, and 30 % of isolates were found to produce strong, moderate, and weak biofilms, respectively. The findings suggest that biofilm forming bacterial strains found in O. pabda fish farm may be a potential source of numerous antibiotic-resistant bacteria. The study sheds new light on antibiotic resistance genes, which are typically inherited by bacteria and play an important role in developing effective treatments or control strategies.

Antibiotic resistance and its correlation with biofilm formation and virulence genes in Klebsiella pneumoniae isolated from wounds.

Klebsiella pneumoniae is the most important species of the Klebsiella genus and often causes hospital infections. These bacteria have a high resistance to most of the available drugs, which has caused concern all over the world. In this study, we investigated the antibiotic resistance profile and the ability to produce extended-spectrum beta-lactamase (ESBL) among K. pneumoniae isolates, and then we investigated the relationship between these two factors with biofilm formation and the prevalence of different virulence genes. In this study, 130 isolates of K. pneumoniae isolated from wounds were investigated. The antibiotic resistance of the isolates was evaluated by the disk diffusion method. The microtiter plate method was used to measure biofilm formation. The prevalence of virulence genes was detected by multiplex PCR. Among the examined isolates, 85.3% showed multidrug resistance. 87.6% of the isolates were ESBL-positive. Imipenem, meropenem, and fosfomycin were the most effective drugs. The ability of the isolates to produce biofilm was strong (80%), moderate (12.3%), and weak (7.6%), respectively. fimH, mrKD, entB, and tolC virulence genes were observed in all isolates. High prevalence of antibiotic resistance (especially multidrug resistance), high prevalence of ESBL-producing isolates, the ability of all isolates to biofilm formation, and the presence of fimH, mrKD, entB, and tolC virulence genes in all isolates show the importance of these factors in the pathogenesis of K. pneumoniae isolates in Iraq.

Altered 16S rRNA gene size in Agrobacterium tumefaciens at IC50 of heavy metals

Agrobacterium tumefaciens is a soil-endemic bacterial species that causes crown galls disease on a range of medicinal and economically important plants. In this study, the diagnosis of this bacterium was confirmed by its formation of crown galls on the discs of carot. And when treated in a laboratory with eight different heavy metals (Mo, Fe, Zn, Cu, Cr, Cd, Ni, and Pb), and with ten concentrations (40, 20, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078 mM/ Metal), it was found to have an inhibitory effect on the growth of these bacteria by microtiter plate method, especially when the concentration was increased. The size of the 16S rRNA gene in bacteria resistant to these metals varied at the half inhibitory concentration IC50 from the comparison sample with volume values of about 1500, 1000, 1000, 1500, 1500 , 1500, 1500, 1500, and 1000 base pairs, respectively.

Antibacterial and antibiofilm potentials of vancomycin-loaded niosomal drug delivery system against methicillin-resistant Staphylococcus aureus (MRSA) infections

The threat of methicillin-resistant Staphylococcus aureus (MRSA) is increasing worldwide, making it significantly necessary to discover a novel way of dealing with related infections. The quick spread of MRSA isolates among infected individuals has heightened public health concerns and significantly limited treatment options. Vancomycin (VAN) can be applied to treat severe MRSA infections, and the indiscriminate administration of this antimicrobial agent has caused several concerns in medical settings. Owing to several advantageous characteristics, a niosomal drug delivery system may increase the potential of loaded antimicrobial agents. This work aims to examine the antibacterial and anti-biofilm properties of VAN-niosome against MRSA clinical isolates with emphasis on cytotoxicity and stability studies. Furthermore, we aim to suggest an effective approach against MRSA infections by investigating the inhibitory effect of formulated niosome on the expression of the biofilm-associated gene (icaR). The thin-film hydration approach was used to prepare the niosome (Tween 60, Span 60, and cholesterol), and field emission scanning electron microscopy (FE-SEM), an in vitro drug release, dynamic light scattering (DLS), and entrapment efficiency (EE%) were used to investigate the physicochemical properties. The physical stability of VAN-niosome, including hydrodynamic size, polydispersity index (PDI), and EE%, was analyzed for a 30-day storage time at 4 °C and 25 °C. In addition, the human foreskin fibroblast (HFF) cell line was used to evaluate the cytotoxic effect of synthesized niosome. Moreover, minimum inhibitory and bactericidal concentrations (MICs/MBCs) were applied to assess the antibacterial properties of niosomal VAN formulation. Also, the antibiofilm potential of VAN-niosome was investigated by microtiter plate (MTP) and real-time PCR methods. The FE-SEM result revealed that synthesized VAN-niosome had a spherical morphology. The hydrodynamic size and PDI of VAN-niosome reported by the DLS method were 201.2 nm and 0.301, respectively. Also, the surface zeta charge of the prepared niosome was − 35.4 mV, and the EE% ranged between 58.9 and 62.5%. Moreover, in vitro release study revealed a sustained-release profile for synthesized niosomal formulation. Our study showed that VAN-niosome had acceptable stability during a 30-day storage time. Additionally, the VAN-niosome had stronger antibacterial and anti-biofilm properties against MRSA clinical isolates compared with free VAN. In conclusion, the result of our study demonstrated that niosomal VAN could be promising as a successful drug delivery system due to sustained drug release, negligible toxicity, and high encapsulation capacity. Also, the antibacterial and anti-biofilm studies showed the high capacity of VAN-niosome against MRSA clinical isolates. Furthermore, the results of real-time PCR exhibited that VAN-niosome could be proposed as a powerful strategy against MRSA biofilm via down-regulation of icaR gene expression.

INVESTIGATION INTO THE IMPACT OF CARVCROL EXTRACTS ON BACTERIA ISOLATES FROM OSTEOMYLITIS AFTER SURGERY

Carvacrol is one of the most ancient medicinal herbs. Carvacrol contains different alkaloids, flavonoids and saponins. In present studies the antimicrobial activity of Carvacrol alcoholic extract against Gram-negative bacterial and Gram-positive bacterial were determined by the microtiter plate method. Gram-negative bacterial and Gram-positive bacterial strains isolated from osteomyelitis patients after surgery in Mosul city , A total of 60 samples were collected from various sources in Mosul hospitals, during the period from January 10, 2024 , to May 1, 2024. The Aim of this study is determining the effectiveness and effect of alcoholic extract of carvacrol on bacterial isolates taken from a Osteomyelitis after surgery. Bacterial culture results on blood agar, MacConkey agar and nutreint agar revealed 54 positive samples for bacterial growth. Among these, Staphylococcus aureus 22 isolates (32.8%), Pseudomonas aeruginosa 12 isolates (17.9%), Enterobacter cloacae 5 isolates (7.5%) Klebsiella pneumoniae 4 isolates (5.9%), Escherichia coli 4 isolates (5.9%), Proteus mirabilis 3 isolates (4.5%), Streptococcus viridans 3 isolates (4.5%), Corynebacterium spp 2 isolates (3%). As for the following bacterial isolates, each of them constituted one isolate out of a total of 67 isolates, at a rate of 1.5% for each: Providencia spp. , Acinetobacter spp., Staphylococcus saprophyticus , Klebsiella oxytoca , Enterococcus spp. , Enterococcus faecium , Citrobacter freundii , Streptococcus groups C , Bacillus cereus , Enterobacter spp. , Acinetobacter baumannii , Morganella morganii, using vitek-2. High-performance liquid chromatography (HPLC) analysis of Oregano plant revealed the presence of active compounds such as Carvacrol, thymol. Plant extracts from carvacrol demonstrated a substantial inhibitory effect. Results: study indicated that plant extracts exhibited higher inhibition than antibiotics. The minimum inhibitory concentration (MIC) while the results of the lowest inhibitory concentration of carvacrol for the bacterial species under study showed that it had an effect at a concentration of 615 µg/ml against Escherichia coli, 307.5 µg/ml for the species Corynebacterium stratium, Morganella morganii and Pseudomonas aeruginosa, and the least effect was 76.875 µg/ml on Staphylococcus aureus, it gave less results than the antibiotic used Carvacrol (Absorbance was measured at Wavelength 600) .

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.

Food Additive Mediated Biosynthesis of AgNPs with Antimicrobial Activity Against Hypermucoviscous Enterotoxigenic Foodborne Klebsiella pneumoniae

This study was aimed to adopt an ecofriendly method to synthesize nanoparticles with an effective antibacterial and anti-biofilm activity. A food origin hypermucoviscous Klebsiella pneumoniae was isolated from food samples and identified using biochemical tests and the Vitek system. A string test was depended on to identify hypermucoviscous isolates. Enterotoxicity of foodborne hypermucoviscous K. pneumoniae isolates was detected phenotypically using suckling mouse bioassay. Biofilm forming ability was tested for all the isolated bacteria using the Microtiter plate method and Congo Red Agar. A natural food additive Syzygium aromaticum (clove) aqueous extract was used for the biosynthesis of silver nanoparticles in optimized conditions. The biosynthesized clove-silver nanoparticles (Clove-AgNps) were characterized by several techniques, and their antimicrobial and antibiofilm activity was determined. The results of this study revealed that the isolation of 28 K. pneumoniae isolates were isolated from 200 food samples. String test results showed that 16/28 (57.14%) K. pneumoniae isolates were hypermucoviscous. Eleven of these isolates (68.75%) were enterotoxigenic. Using clove aqueous extract as a biological agent was successful in the biosynthesis of AgNPs with an average diameter of 14.12 nm as measured by AFM. The optimum biosynthesis conditions were: 1mM of AgNO3 concentration, pH 7, at 37 °C, and 24 hours. The minimum inhibitory concentration of the clove-AgNPs was detected as 62.5 µg.mL-1. Sub-inhibitory concentration of 31.25 µg.mL-1 of Clove-AgNPs resulted in: 91% decrease in the formed biofilm. It can be concluded that using Syzygium aromaticum is an effective ecofriendly method for AgNPs biosynthesis with excellent antibacterial and anti-biofilm activity against enterotoxigenic hypermucoviscous K. pneumoniae.

Study of MIC of silver and zinc oxide nanoparticles, strong and cost-effective antibacterial against biofilm-producing Acinetobacter baumannii in Shiraz, Southwest of Iran

BackgroundAcinetobacter baumannii resistant strains lead to increased mortality, treatment costs, and an increase in the length of hospitalization. Nowadays, nanoparticles are considered a substitute for antibiotics. This study aimed to determine the MIC of Silver (Ag) and Zinc Oxide (ZnO) Nanoparticles (NPs) on Biofilm-Producing Acinetobacter baumannii and determine the relationship between MIC and frequency of efflux pump genes in cutaneous specimens in Shiraz, Southwest Iran in 2021–2022.MethodsIn this study, specimens were collected from April 2021 to June 2022 at Namazi and Faqihi Hospitals in Shiraz. Investigation of biofilm production in multidrug resistance (MDR) isolates was done by the microtiter plate method. Synthesized nanoparticles were characterized by UV–vis spectrum, X-ray diffraction (XRD), and electron microscopy. The MIC of AgNPs and ZnONPs for isolates was done using the method described in the CLSI guideline (2018). The antibacterial effect of MIC of NPs on inanimate objects was done by colony counts. The prevalence of efflux pump genes (adeR, adeC, adeA, abeM, adeK, adeI) was also investigated by PCR technique.ResultsThe highest ceftriaxone resistance (68%) and lowest colistin resistance (7%) were identified. 57% of isolates were MDR. In addition, 71.9% could produce biofilm and 28.1% of isolates could not produce biofilm. The average size of AgNPs and ZnONPs in the present study is 48 and < 70 nm, respectively. The nanoparticles were spherical. The MIC and the MBC of the ZnONPs were in the range of 125 to 250 µg/mL respectively. Also, for AgNPs, the MIC and the MBC were in the range of 62.5 to 250 µg/ml, respectively. AbeM gene had the highest frequency and the AdeK gene had the lowest frequency. Statistical analysis showed that there is a relationship between the frequency of adeA, adeC, and adeM genes with the MIC of AgNPs and ZnONPs.ConclusionAccording to the results of the present study, inanimate objects such as scalpels in contact with AgNPs (6000 µg/ml for 240 min) or ZnONPs (5000 µg/ml for 120 min) can be free of biofilm producing Acinetobacter baumannii with efflux pump genes.

Multiple-Locus Variable-Number Tandem Repeat Analysis Genotyping of Biofilm-Producing Pseudomonas aeruginosa Clinical Isolates

Background: Pseudomonas aeruginosa (P. aeruginosa) significantly contributes to hospital-acquired infections. Objectives: This study aimed to investigate the genetic diversity of P. aeruginosa strains using multiple-locus variable-number tandem repeat analysis (MLVA) and to explore the relationship between biofilm production and antibiotic resistance. Methods: In this cross-sectional study, 79 P. aeruginosa isolates were collected. Antibiotic sensitivity was tested using the Kirby-Bauer method, and biofilm production capability was assessed through the microtiter plate method. Genetic diversity was evaluated by MLVA, analyzing eight variable-number tandem repeat (VNTR) loci: MS-213, MS-214, MS-207, MS-217, MS-222, MS-209, MS-77, and MS-172. Phylogenetic relationships were delineated using PHYLOViZ 2.0 software. Results: The patient cohort comprised 51.9% males, with the majority of samples (35.4%) obtained from urine. Ceftazidime (CAZ 30µg) showed the highest resistance rate at 77.2%. Notably, 92.4% of isolates were capable of forming biofilms, categorized as 22.7% weak, 28.7% moderate, and 46.5% strong. Phylogenetic analysis demonstrated variability across one or more VNTR loci. Simpson’s index (0.906) and Shannon-Weiner diversity indices (H: 3.466, J: 0.910, Hmax: 3.807, Hmin: 1.242) identified MS77 as the most informative marker for genetic diversity among the isolates. Conclusions: The study highlights an alarming trend in antibiotic resistance, underscoring the necessity of regular monitoring. The findings confirm that MLVA is a straightforward, rapid genotyping method suitable for assessing the genetic diversity of P. aeruginosa.

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.

Molecular diversity of Klebsiella pneumoniae clinical isolates: antimicrobial resistance, virulence, and biofilm formation

One of the mechanisms responsible for antibiotic resistance in Klebsiella pneumoniae is the enzymes produced by the bacteria; another important mechanism is the ability to form biofilm. In this study, antibiotic resistance, genes associated with virulence, and biofilm-forming properties of K. pneumoniae strains were investigated. A total of 100 K. pneumoniae isolates were obtained from different clinical samples identified by Matrix-Assisted Laser Desorption/Ionization time-of-flight Mass Spectrometry. Antimicrobial susceptibility testing was performed with the Phoenix 100 apparatus. The biofilm forming properties of strains were determined by the microtiter plate method. For molecular analysis, genes encoding the carbapenemase enzyme (bla OXA-48, bla NDM-1, bla IMP, and bla VIM) and biofilm-related genes (treC, luxS, mrkA, and wza) were investigated by polymerase chain reaction (PCR). While 76% of clinical isolates were resistant to three or more antimicrobials, 24% were classified as non-multidrug resistant (non-MDR). When biofilm-forming capacities of clinical isolates were tested, it was determined that the resistant-isolates produced 59.2% strong biofilm, and susceptible-isolates produced 12.5% strong biofilm. According to PCR results, carbapenemase genes were determined as follows: bla OXA−48-70%, bla NDM-49%, and bla KPC-19%, bla OXA-48/bla NDM/bla KPC-12%, bla OXA-48/bla NDM-26%, and bla OXA-48/bla KPC-4%. The biofilm-associated genes in bacterial isolates were determined as follows: luxS-98%, treC-94%, mrkA-88%, and wza-15%. In addition, Hierarchical Clustering Tree and Heatmap analysis revealed an association between isolates that lacks resistance genes and isolates lacks biofilm-formation related genes that were included in MDR or non-MDR classes. As a result, biofilm should be considered in the treatment of MDR infections, and therapy should be planned accordingly. In addition, pursuing the data and genes of antibiotic resistance is significant for combating resistance.

Treatment and Prevention of Oral Biofilms by Clove _Gold Nanoparticles (Comparative Study).

The goal of the study was to find out the antibiofilm activity of biosynthesizing gold nanoparticles derived from clove buds extracts against certain oral bacteria, to compare between preventive and treatment protocols. The antibiofilm activity was tested using the Microtiter plate method against oral bacteria (Enterococcus faecalis, Streptococcus mutans and lactobacillus spp). This study has showed a good antibiofilm. effect on oral bacteria.

Phenotypic and Genotypic Virulence Potentials of Listeria monocytogenes Isolated from Different Food Samples in Yola

Aim: the aim of this work is to determine the Phenotypic and Genotypic Virulence Potentials of Listeria monocytogenes Isolated from Different Food Samples in Yola. Study Design: the study was a randomized study; the samples (sources of organisms) were collected at random from different market and shops within the study area. Place and Duration of Study: the study was carried out in Yola, the capital of Adamawa State, north eastern Nigeria. Methodology: phenotypic virulence potentials of the isolates was determined by haemolytic activity on 5% sheep blood agar and biofilm formation ability, using Christensen’s tube method and microtiter plate method for the qualitative and quantitative biofilm formation respectively. Genotypic virulence determination was done by polymerase chain reaction amplification of plcB, hlyA and actA genes. Results: All the isolates (100%) were beta haemolytic on 5% sheep blood agar and all the isolates were biofilm formers but with different degree of biofilm formation abilities. Genotypically all the isolates (100%) indicated the presence of all the three virulence genes amplified. Conclusion: food samples isolates analyzed are potentially virulence pathogenic of L. monocytogenes that can pose food borne listeriosis to high risk individuals in the study area.

Antibiotic Resistance and Biofilm Formation in Gram Negative Bacteria Isolated From Endotracheal Tubes in Intensive Care Unit of a Tertiary Care Hospital

Biofilm formation has been found to play a central role in many human infections in which antibiotic resistance is common. Bacteria can colonize and form biofilms on almost any surface, including natural and synthetic surfaces the present study was conducted with an aim to detect biofilm formation in ET tubes/ ET secretions received from ICU patients by microtiter plate method. In the present study a total of 85 isolates were obtained out of 400 samples. Among 85 isolates of gram- negative bacteria, maximum number of isolates were of K. pneumoniae (41.17%) followed by A. baumannii (40%), P. aeruginosa (10.5%), E. coli (5.88%) and B.cepacia (2.35%). All isolates were tested for antimicrobial sensitivity/resistance by Vitek 2 Compact system. All the isolates were further tested for biofilm production by microtiter plate method and optical density (OD value) was measured. Biofilm production was distributed by strong, medium, and weak. It is concluded that stringent hygiene protocols, routine tube changes and the use of antimicrobial coatings are crucial for prevention of biofilm development in ICU settings.

In vitro investigation of relationship between quorum-sensing system genes, biofilm forming ability, and drug resistance in clinical isolates of Pseudomonas aeruginosa

BackgroundPseudomonas aeruginosa is an opportunistic pathogen in the health-care systems and one of the primary causative agents with high mortality in hospitalized patients, particularly immunocompromised. The limitation of effective antibiotic administration in multidrug-resistant and extensively drug-resistant P. aeruginosa isolates leads to the development of nosocomial infections and health problems. Quorum sensing system contributes to biofilm formation, expression of bacterial virulence factors, and development of drug resistance, causing prolonged patient infections. Therefore, due to the significance of the quorum sensing system in increasing the pathogenicity of P. aeruginosa, the primary objective of our study was to investigate the frequency of quorum sensing genes, as well as the biofilm formation and antibiotic resistance pattern among P. aeruginosa strains.MethodsA total of 120 P. aeruginosa isolates were collected from different clinical specimens. The disk diffusion method was applied to detect the antibiotic resistance pattern of P. aeruginosa strains. Also, the microtiter plate method was carried out to evaluate the biofilm-forming ability of isolates. Finally, the frequency of rhlI, rhlR, lasI, and lasR genes was examined by the polymerase chain reaction method.ResultsIn total, 88.3% P. aeruginosa isolates were found to be multidrug-resistant, of which 30.1% had extensively drug-resistant pattern. The highest and lowest resistance rates were found against ceftazidime (75.0%) and ciprofloxacin (46.6%), respectively. Also, 95.8% of isolates were able to produce biofilm, of which 42.5%, 33.3%, and 20.0% had strong, moderate, and weak biofilm patterns, respectively. The frequency of quorum sensing genes among all examined strains was as follows: rhlI (81.6%), rhlR (90.8%), lasI (89.1%), and lasR (78.3%). The most common type of quorum sensing genes among multidrug-resistant isolates were related to rhlR and lasI genes with 94.3%. Furthermore, rhlI, rhlR, and lasI genes were positive for all extensively drug-resistant isolates. However, the lasR gene had the lowest frequency among both multidrug-resistant (83.0%) and extensively drug-resistant (90.6%) isolates. Moreover, rhlR (94.7%) and lasR (81.7%) genes had the highest and lowest prevalence among biofilm-forming isolates, respectively.ConclusionOur findings disclosed the significantly high prevalence of drug resistance among P. aeruginosa isolates. Also, the quorum sensing system had a significant correlation with biofilm formation and drug resistance, indicating the essential role of this system in the emergence of nosocomial infections caused by P. aeruginosa.