Congo Red Agar Method Research Articles

Emerging threats of high biofilm formation and antibiotic resistance in clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates from Pakistan

ObjectivesThis multicenter study, conducted from a One Health perspective, aimed to comprehensively examine the prevalence of Methicillin-resistant Staphylococcus aureus (MRSA) infections and their biofilm-forming capabilities in Pakistan. Phylogenetic analysis of the study isolates was also performed. MethodsA total of 150 MRSA isolates were screened from various clinical samples using Cefoxitin antibiotic discs. Genotypic confirmation was conducted through mecA, S. aureus-specific nuc, and 16S rRNA genes. Biofilm formation was assessed using Congo red agar (CRA) and slime layer quantification methods. The intercellular adhesion (ica) operon genes, specifically icaA and icaD, were detected. Phylogenetic analysis utilized the 16S rRNA sequences. Statistical associations between various parameters were determined using chi-square analysis. ResultsThe presence of the mecA gene was observed in 131 out of 150 isolates (87.3%). CRA identified 28% and 40% of isolates as strong and moderate biofilm producers, respectively, while 9.3% were classified as non-biofilm producers. The slime layer assay exhibited higher sensitivity, classifying only 4.7% of isolates as non-biofilm producers. Biofilm-forming genes icaA and icaD were detected in 85.3% and 86.7% of the isolates, respectively. Antibiotic resistance was more prevalent among biofilm-forming isolates, particularly against ciprofloxacin, levofloxacin, erythromycin, trimethoprim-sulfamethoxazole, and fosfomycin. Ceftaroline demonstrated efficacy irrespective of biofilm-forming abilities. Conversely, non-biofilm producers exhibited complete susceptibility to clarithromycin and tigecycline. ConclusionsClinical MRSA strains exhibit a substantial potential for biofilm formation, contributing to a resistant phenotype. Routine antibiotic testing in clinical settings that overlook the biofilm aspect may lead to the failure of empiric antibiotic therapy.

ZnO nanoparticles induced biofilm formation in Klebsiella pneumoniae and Staphylococcus aureus at sub-inhibitory concentrations.

Biofilm formation by the pathogenic bacteria generates a serious threat to the public health as it can increase the virulence potential, resistance to drugs, and escape from the host immune response mechanisms. Among the environmental factors that influence thebiofilm formation, there are only limited reports available on the role of antimicrobial agents. During the antimicrobial drug administration or application for any purpose, the microbial population can expect to get exposed to the sub-minimum inhibitory concentration (sub-MIC) of the drug which will have an unprecedented impact on microbial responses. Hence, the study has been conducted to investigate the effects of sub-MIC levels of zinc oxide nanoparticles (ZnO NPs) on the biofilm formation of Klebsiella pneumoniae and Staphylococcus aureus. Here, the selected bacteria were primarily screened for thebiofilm formation by using the Congo red agar method, and their susceptibility to ZnO NPs was also evaluated. Quantitative difference in biofilm formation by the selected organisms in the presence of ZnO NPs at the sub-MIC level was further carried out by using the microtiter plate-crystal violet assay. Further, the samples were subjected to atomic force microscopy (AFM) analysis to evaluate the properties and pattern of thebiofilm modulated under the experimental conditions used. From these, the organisms treated with sub-MIC levels of ZnO NPs were found to have enhanced biofilm formation when compared with the untreated sample. Also, no microbial growth could be observed for the samples treated with the minimum inhibitory concentration (MIC) of ZnO NPs. The results observed in the study provide key insights into the impact of nanomaterials on clinically important microorganisms which demands critical thinking on the antimicrobial use of nanomaterials.

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. 

Antibacterial and antibiofilm activities of Thymus leptobotrys and Lavandula mairei essential oils against extended spectrum β-lactamase producing Enterobacteriaceae

The aim of the study was to investigate the antibacterial and antibiofilm activities of Lavandula mairei and Thymus leptobotrys essential oils against Extended spectrum β-lactamase producing Enterobacteriaceae, responsible of nosocomial infections. PCR and sequencing methods detected three different resistance genes (blaSHV-42, blaSHV-12 et blaCTXM-15) in the studied strains. Gas chromatography-mass spectrometry analysis highlighted the predominance of carvacrol in both Lavandula mairei and Thymus leptobotrys essential oils. Their antibacterial activity was conducted by disk diffusion and microdilution methods. Great sensitivity of the multidrug resistant strains was noted for the two essential oils with inhibition zones’ diameters ranging from 13.3±2.3 to 32.3±1.4 mm (Thymus leptobotrys) and from 22 to 34.66±0.57 mm (Lavandula mairei), and low minimal and bactericidal concentrations varying from 1.56 to 6.25 mg/mL (CMI) and from 1.56 to 12.5 mg/mL (CMB) for both essential oils. Four strains out of six, considered as the strongest biofilm producers by congo red agar and crystal violet methods, were chosen to evaluate biofilm inhibition and eradication effects of Lavandula mairei and Thymus leptobotrys essential oils. In most cases, these latter inhibited considerably the biofilm formation of the targeted strains with proportions greater than 80%. The eradication effect was lower, with rates not higher than 50%. Thus, these essential oils could be used as an effective alternative approach to prevent biofilm formation of multidrug resistant Enterobacteriaceae. To our knowledge, no previous works have reported the antibiofilm activity of Lavandula mairei and Thymus leptobotrys essential oils against these multidrug resistant bacteria.

Comparison of antimicrobial efficacy of different disinfectants on the biofilm formation in dental unit water systems using dip slide and conventional methods: A pilot study.

Biofilm formation in dental waterlines brings opportunistic infections, especially for immunosuppressive patients. This study aimed to determine biofilm-forming microorganisms by various methods and investigate disinfectants' effects on biofilm. In the study, samples were obtained from the waterlines of 10-15 aged six dental units, before (0 min.) and after chlorine dioxide (ClO2 ) and hypochlorous acid (HOCl) treatment (1, 5, 10, 20, and 30 min.), and total colony counts were performed using conventional surface smear method (SSM) and dip slide method (DSM). The Congo red agar and Christensen methods were used to examine the biofilm-forming properties of the isolates. Monitoring of biofilm presence was also visualized by SEM scanning. When DSM and SSM are compared in all units where ClO2 and HOCl are applied, DSM can detect bacterial growth even during periods of greater exposure to disinfectant application. Although DSM can achieve a value approaching 3% even at the 10th minute in units treated with HOCl; SSM does not show reproduction at the same disinfectant exposure and duration; It was observed that in the units where ClO2 was applied, the growth was no longer observed at the 10th minute with DSM, and SSM, 50% growth in the first minute of the units treated with ClO2 could not be detected in the 5th minute. It is concluded that it can be advisable to routinely disinfect the dental unit water systems with non-toxic doses of ClO2 application before patient treatments in clinics and also to perform contamination controls at regular intervals with DSM, which is a sensitive and very practical method. It has been observed that the dip slide method can count bacteria more sensitively than conventional methods in dental water systems without the need for experienced personnel and equipment. The difference between biofilm formation in water systems before and after disinfectant exposure in SEM examinations is remarkable. The effects of ClO2 and HOCl on biofilm were investigated and bacterial growth was inhibited in dental units between 5 and 10 minutes with both disinfectants.

Study of biofilm formation among uropathogens isolated from catheter-associated UTI patient from a tertiary care hospital

Background: Catheter-associated urinary tract infection (CAUTI) is a serious health threat and challenging infection. CAUTI accounts for up to 40% of all nosocomial infections. Biofilm provides a survival strategy to microorganisms and ultimately leads to re-infections and recurrence of urinary tract infections (UTI) despite a full course of antibiotics. Aims and Objectives: The present study was conducted to determine the prevalence of CAUTI in suspected UTI patients and prevalence of biofilm-forming uropathogens among CAUTI patients. Materials and Methods: The cross-sectional study was done over a period of 6 months among 95 catheterized CAUTI patients. Biofilm production among isolated uropathogens was tested by tissue culture plate, tube test, and congo red agar method. Isolates were identified as biofilm producer if they were tested positive by any one of the all four methods, and isolates were considered biofilm non-producer in consensus with all four methods. Results: In this study, the prevalence of CAUTI was 68.42%. Among 65 isolates most common uropathogen was 28 (43.07%) Escherichia coli. In this present study, the prevalence of biofilm-forming uropathogens was 58.46% (38). Tissue culture plate was the most sensitive (97.36%) method in detecting biofilm formation followed by modified congo red agar (82.21%), congo red agar (71.05%), and tube test (65.78%). Biofilm productions were significantly associated with female gender, diabetes, and prolonged catheterization. Conclusion: Indwelling urinary catheter acts as a nidus for biofilm formation among microorganisms. Duration of catheterization is inversely associated with UTI. Hence, the need for catheter removal should be assessed daily to prevent infection. Periodic surveillance should be done to detect biofilm formation where prolonged catheterization is inevitable.

Staphylococcus Aureus and Pseudomonas Aeruginosa Biofilms in Ear-Set Associated Infection

Background and objectives: Ear infections are a serious public health issue in developing countries. Gram-positive and gram-negative Bacteria are both capable of developing biofilms on medical equipment and earphones; however, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Enterococcus faecalis are the most prevalent types and may involve in ear infections. Since there was no such study in our region about ear set association with biofilm formation in patients with ear infections, this study was accomplished. The aim of the study: Therefore, this study was suggested in order to evaluate the relationship between the continuous use of Ear set with biofilm formation and ear infections among handlers. Patients, Materials and Methods: This study was carried out in Kirkuk City (The urban region only) from the 15th of January 2023 to the 23rd of May 2023, done on a total number of (168) participants. The total number of the patients who were Ear set users enrolled for the current study was (120), divided into two groups including (60 patients with biofilm positive (including 27 male and 33 female patients respectively) and 60 patients with biofilm negative (including 23 male and 37 female patients separately), with age, ranged from 20 to 60 years old, who were attended at Kirkuk Teaching Hospital. The control group (non-Ear set users) of the presented study was a total number of (48) including (18) males and (30) females. The serological tests included: the estimation of Human GR, SOD, and MDA antioxidants levels through using of the ELISA technique for all the participants, and the biochemical tests included measuring Vitamin D3 levels by using the iChromaTM technique, while the microbial tests were done through using of VITEK 2 compact system. Biofilm formation was detected by using the Congo red agar method. All the steps of methods are conducted based on the manufacturer's instructions. The bacterial growth among the patients group was (22.5%) of Staphylococcus aureus and (14.16%) of Pseudomonas aeruginosa, while the control group revealed (8.33 %) of Staphylococcus aureus and (4.16%) of Pseudomonas aeruginosa. The age range (20-30) years showed the highest rates among the infected patients, with male patients being more vulnerable to the infections. The mean levels of vitamin D3, human GR, SOD, and MDA decreased significantly in patients in matching to control groups. Conclusions: As a conclusion, Staphylococcus aureus and Pseudomonas aeruginosa are more prevalent agents involved in ear infections. Male patients are more susceptible patients to contracting ear infections. Vitamin D3, Human GR, SOD, and MDA antioxidants levels decline in patients with ear infections.

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.

Antibiotic Resistance Profile of Biofilm Producing Staphylococci Isolated from Different Clinical Samples

Biofilm mediated infections by Staphylococci have a significant negative impact on patient health and necessitate reliable method for detecting biofilm producers. The ability of isolates to produce biofilm makes them resistant to host immune response as well as available antibiotics. This study aims to detect biofilm producing ability among clinical staphylococci by phenotypic methods and presence of icaAD genes and determine their antibiotic profile. A total of 350 different clinical specimens received in the KIST Medical hospital, Gwarko, Nepal was examined from July 2018 to January 2019 and Staphylococci were identified following standard microbiological procedure. The antibiotic resistivity pattern was detected by Kirby Bauer disc diffusion method whereas biofilm formation was detected by three phenotypic methods viz. congo red agar (CRA), tube method (TM) and tissue culture plate (TCP) method. Furthermore, icaAD genes were detected by PCR method. Chi-square test and independent sample t-test were employed to calculate the significance. A total of 161 Staphylococci were isolated comprising S. aureus (63, 39.1%) and coagulase negative staphylococci (CNS) (98, 60.9%). Most isolates were found to be resistant to penicillin and erythromycin. Strong biofilm formation was detected among 6 (3.7%), 22 (13.7%) and 35 (21.7%) by CRA, TM and TCP method respectively. Similarly, icaAD genes were detected among 24 (14.9%) isolates. Staphylococci isolated from clinical sample showed biofilm forming ability by both phenotypic and genotypic method. The biofilm producing isolates were found to be more resistant to antibiotics than their planktonic counterparts with 56 (89%) and 65 (66%) S. aureus and CNS observed as methicillin resistant respectively. Regular surveillance of biofilm formation by Staphylococci and their antimicrobial resistance profile may lead to early treatment of Staphylococcal infection.

Comparative Study between Ear Set Use in Relation with Bacterial Biofilms Causing Ear-Infections

Ear infections were recently considered as a serious public health issue in many countries; where Gram-positive and Gram-negative bacteria are the most associated organisms. Besides, the ability of many species of those microorganisms to grow biofilms on medical equipments and different head set raised the question about the relationship between widespread use of ear set and complicated ear infections. Therefore, this study was suggested in order to evaluate the relationship between the continuous use of ear set with biofilm formation and ear infections among handlers. Patients, Materials and Methods: This study was carried out in Kirkuk City during January to May 2023 on a total number of (168) participants from both sexes and age range between 20-60 years. Participants were divided into two groups according to ear set use; Group A included (n=120) headphone users with (50 male and 70 female), who attended Kirkuk Teaching Hospital. On the other hand, Group B included non-headphone users of (n=48) participants with (18 male and 30 female) as the control group. Ear swab samples were collected aseptically under the supervision of Otolaryngologist Consultant from both groups and were immediately cultivated on selective media for microbiological and biochemical diagnostic tests. In accordance, the biofilm formation ability by the isolated bacteria was distinguished using the Congo red agar method. In addition, this study involved certain serological test including: the estimation of Human GR, SOD, and MDA antioxidants levels through using of the ELISA technique for all the participants, and the biochemical tests included measuring Vitamin D3 levels by using the ichromaTM technique to through the light on other factors which might be associated with ear infections. Diagnostic tests revealed the isolation of (28.33%) Pseudomonas aeruginosa, and (45%) of Staphylococcus aureus biofilm positive isolates from ear swabs of Group A participants. While, ear swabs collected from Group B had displayed the isolation of (4.16%) of Pseudomonas aeruginosa, and (8.33 %) of Staphylococcus aureus isolates respectively. Besides, the age range of (20-30) years old showed the highest rates among the infected patients, with male patients being more vulnerable to the infections than their female spouse. Moreover, Vitamin D3, human GR, SOD, and MDA antioxidant markers showed a significant decrease among participants of Group A in comparison to Group B Vitamin D3 and antioxidant marker levels.Staphylococcus aureus and Pseudomonas aeruginosa were the most prevalent agents involved in ear infections. Male patients were more susceptible to contracting ear infections. Vitamin D3, Human GR, SOD, and MDA antioxidant Marker levels declined in patients with ear infections especially the users of headphones.

Detection of Biofilm Production and Antibiotic Resistance Pattern in Clinical Isolates from Orthopaedic Implants Associated Infections

Introduction: Orthopaedic Implant associated infection is the major problem which leads to implant failure and economic burden to the patient. Implant-associated infections are due to the formation of biofilm at the implantation site leading to multidrug resistant organism. Aim: This study was done to evaluate the causative organisms, their antibiotic sensitivity pattern and their ability to form biofilm over the Implants used in Orthopaedic surgeries. Material and methods: This study was carried out in the department of Microbiology, Adichunchanagiri institute of Medical sciences from September 2019 to August 2022. Swabs from 100 patients who had undergone orthopaedic implant or prosthetic surgery and presented with signs and symptoms of infection were collected and processed as per standard procedures. All the isolates were subjected to detect biofilm production by Congo red agar method, MRSA detection by Cefoxitin disc diffusion test, vancomycin resistant Enterococcus by using Vancomycin (Minimum inhibitory concentration) MIC strips and Extended spectrum β lactamases (ESBL) production by phenotypic confirmatory combined-disc test. Results: Out of the 100 samples processed, culture positivity was observed in 72 specimens. Among them, Staphylococcus aureus 19(26%) was the predominant isolate and 44 (61%) are biofilm producers. 17 (89%) were MRSA (Methicillin resistance Staphylococcus aureus) strains and 15(37%) were ESBL producing strains. Conclusion: The appropriate pre- and post- operative care should be taken to prevent orthopaedic implant associated infections. Staphylococcus spp was the commonest isolate and its ability to produce biofilm indicates the need for an appropriate antibiotic policy and screening for MRSA carriers to reduce the infection. Keywords: Orthopaedic implant, Infection, Biofilm, MRSA, ESBL

Presence of methicillin-resistant Staphylococcus aureus in raw cow’s milk: adhesive capacities and extracellular enzymes characterization

Raw milk contamination by Methicillin-resistant S. aureus (MRSA) threatens food safety and leads to public health problems. Our study evaluates biofilm formation and virulence factors among 21 MRSA in raw cow’s milk. Methicillin resistance was confirmed by cefoxitin screening using the automated VITEK2 system, with a minimum inhibitory concentration greater than 8 mg/l. Qualitative characterization of biofilm-producing strains was performed on Congo red agar (CRA), with a semi-quantitative adhesion test on 96-well tissue culture plates (TCP). The ability to produce different enzymes was evaluated, such as caseinase, lipase, and phospholipase (lecithinase). The surface hydrophobicity of the bacteria was determined, and the autoaggregation test was used to predict the interactions between bacterial cells. Among the tested strains, 61.9% were biofilm producers in the CRA, developing a positive and variable phenotype. Furthermore, 19.05% and 80.95% of isolates were high and low biofilm formation on TCP. The enzymatic activity showed that lécithinase, caseinase, and lipase activities were detected in 100%, 80.95%, and 80.95% of cases. Highly hydrophilic (85.71%) and weakly hydrophobic (14.29%) were detected in MRSA isolates. The strains showed that 71.43% were moderate autoaggregation and 28.57% were low autoaggregation. No significant difference was found between the CRA method and TCP (P&gt; 0.05). A significant association was found between adhesion capacity and bacterial autoaggregation in S. aureus strains (p&gt;0.05). On the other hand, no statistical association between the hydrophobicity of microbial strains and adhesion capacity (p&lt;0.05) was found. The same result was for the hydrophobicity of microbial strains and autoaggregation (p&lt;0.05). This investigation could be beneficial for developing new control measures, prevention, and effective treatment against staphylococcal bacterial resistance.

ISOLATION AND IDENTIFICATION OF BIOFILM-FORMING BACTERIA IN FOODS SUPPLIED FOR CONSUMPTION IN OPEN-AIR MARKET STALLS: A CASE STUDY IN AYDIN PROVINCE

In this study, the isolation and identification of bacteria that form biofilms on various food products sold on open-air market stalls in Aydın Province were aimed. Bacteria were isolated from food surfaces in aseptic conditions and isolated after incubation in the Tryptic Soy Agar medium at 37°C for 24-48 hours. DNA isolations of the isolated bacteria were performed and the PCR products obtained were used for sequencing. The Congo Red Agar method was used to qualitatively analyze biofilm formation. According to this method, bacteria forming black colonies were evaluated as biofilm-positive and were subjected to quantitative analysis using the Microplate method. A total of 67 bacteria were isolated from the sampled food items, of which 7 were strong and 2 were moderate biofilm producers, showing that more importance should be given to food hygiene.

Prevalence of urinary tract infection and antimicrobial resistance patterns of uropathogens with biofilm forming capacity among outpatients in morogoro, Tanzania: a cross-sectional study

IntroductionUrinary tract infection (UTI) is the second most common infectious disease affecting more than 150 million people globally annually. Uropathogenic E. coli (UPEC), the predominant cause of UTI, can occur as a biofilm associated with antimicrobial resistance (AMR). There is a data gap on global AMR patterns from low-income settings, including Tanzania. Data on antimicrobial susceptibility patterns in relation to biofilm formation will help in the proper selection of antibiotics and the fight against AMR.MethodsThis analytical cross-sectional study was conducted among consecutively selected outpatients (n = 344) from January to May 2022 at Morogoro Regional Referal Hospital. Mid-stream urine samples were collected aseptically from symptomatic patients. A significant UTI was defined when more than 105 colonies/ml of urine were recorded. Kirby Bauer’s disc diffusion method was used for antibiotics susceptibility patterns and a Congo Red Agar method was used to determine biofilm formation. Two-sided χ2 test or Fisher’s exact test, Cohen’s kappa coefficient and logistic regression were used for data analysis. A p-value < 0.05 was considered statistically significant.ResultsThe prevalence of UTIs was 41% (141/344) and elders (>=60 years) had five times higher odds of having UTI as compared to adolescents (p < 0.001). E. coli was the most predominant bacteria (47%; 66/141), which displayed moderate susceptibility against ciprofloxacin (59.1%) and nitrofurantoin (57.6%). A total of 72 (51%) of all isolated bacteria were multi-drug resistant. All isolated bacteria demonstrated high resistance (> 85%) against ampicillin and co-trimoxazole. In this study, 51.5% (34/66) were biofilm-forming E. coli and demonstrated relatively higher antibiotic resistance as compared to non-biofilm forming bacteria (p < 0.05).ConclusionWe report high antibiotic resistance against commonly used antibiotics. Slightly more than half of the isolated bacteria were biofilm forming E. coli. A need to strengthen stewardship programs is urgently advocated.

ASSESSMENT OF ANTIBIOTICS RESISTANCE AND BIOFILM PRODUCTION AMONG BACTERIAL SPECIES ISOLATED FROM CONTACT LENSES

Contact lenses (CLs) wearing has been increased globally during recent decades, which is one of the main risk factors for developing several ocular infections. Resistant CLs bacterial infections are mainly due to the CLs contamination by bacteria producing biofilm. This study was aimed to assessment of antibiotics resistance and biofilm production among bacterial species isolated from contact lenses in Mukalla city, Hadhramout, Yemen. This cross-sectional study was carried out on 298 participants women during a period from October 2022 to January 2023. The CLs swab samples were collected, then inoculated onto culture media and incubated aerobically at 37°C for 24 hrs. The bacterial isolates were identified by conventional bacteriological methods of cultural characteristics, Gram staining and biochemical test. Antibiotics susceptibility testing was performed by disc diffusion method. Bacterial biofilm production on CLs was detected by tube method (TM) and Congo red agar (CRA) method. The prevalence of CLs bacterial infection was 54.4%. Enterobacter spp. 37.1%, followed by Escherichia coli 28.4%, Pseudomonas aeruginosa 11.7%, Klebsiella pneumoniae 6.8% were the most common Gram-negative isolated from CLs. Staphylococcus epidermides 3.7% and other coagulase negative staphylococci (CoNS) 12.3% were the most common species of Gram-positive bacteria isolated from CLs. The CRA method was found to be effective phenotypic screening method for detection of biofilm production of bacterial isolates from CLs. Prevalence of antibiotics resistance and multi-drug resistance (MDR) biofilm producing strains was found. In conclusion, there is a high prevalence of CLs use by females in Mukalla city, Hadhramout especially for cosmetic purposes. CLs infection due to improper care practices leads to eye complications. Increasing awareness is crucial to avoid identified risk factors for ocular infection.

Prevalence Study of Biofilm Marker ica D Gene among Coagulase Positive Staphylococcus of Mastitic Origin

Bovine Mastitis is an inflammation of the mammary gland, usually due to microbial infection, which causes economic loss to dairy producer every year. Staphylococcus is one of the most common pathogens responsible for contagious mastitis in bovines. The aim of this study was to identify coagulase positive Staphylococci isolated from bovine subclinical mastitis. In vitro biofilm forming ability of the isolates, an important virulence factor of mastitogen and prevalence of biofilm marker icaD gene among the isolates. In the present study, 70 isolates were biochemically identified as S. aureus. Biochemically characterized strains of S. aureus were screened for biofilm formation. Phenotypic detection of biofilm formation of S.aureus were done by two methods, namely, Congo Red Agar (CRA) method and Tube Method (TM). The frequency of biofilm formation in S. aureus strains was 28.57 per cent on CRA plates while 71.42 per cent were biofilm non-producers. The frequency of biofilm formation in S. aureus strains was 38.57 per cent in total as shown by tube method while 61.42 per cent were biofilm non-producers. Employing PCR, molecular detection technique of icaD gene was carried out. In this study, 72.85 per cent S. aureus strains were found to be positive for icaD gene which produced an amplicon of 381bp.

Virulence Factors of Clinical and Fecal Isolates of Enterococci Species

Enterococci species are known commensals of the gastrointestinal flora; however, in recent years, they have emerged as important nosocomial pathogens that possess many virulence factors that are attributed to the pathogenesis of diseases caused by them. The study evaluated and compared the virulence factors of Enterococci isolated from fecal and clinical samples. From the obtained isolates, the clinical enterococcal isolates produced 35%, 20%, and 50%, and fecal isolates produced 23%, 13%, and 13% gelatinase, hemolysin, and biofilm, respectively. Biofilm production determined by the Congo Red agar, tube, and microtiter plate methods was 23%, 39%, and 49%, respectively. The sensitivity of the Congo Red agar and tube method compared to the microtiter plate method was 27% and 46%, respectively, whereas the specificity of both tests was 79%. This study showed that biofilm production plays a significant role in the pathogenesis of diseases caused by Enterococci. Detection of biofilm production using the microtiter plate method is more sensitive and specific than the Congo Red agar and tube method.

Detection of biofilm formation in multi-drug resistant Staphylococcus aureus among clinical isolates in Zaria metropolis, Kaduna, Nigeria

Antimicrobial resistance are associated with several virulence factors such as biofilm. The biofilm formation ability of S. aureus complicates effective therapeutic management of infections. In this research, we intend to observe biofilm formation in S. aureus using two in vitro phenotypic methods: tissue culture plate and Congo red agar as well as biofilm associated genes Ica A B &amp; D and 16SrRNA. A total of 150 presumptive coagulase positive staphylococcal isolates from all specimens (blood, urine, high vaginal swab, wound swab, ear swab, endocervical swab) submitted to the microbiology laboratory of the selected hospitals were collected. Seventy-three (73) or 48.7% of the isolates were identified as coagulase positive Staphylococcus using microbiology standard methods (tube coagulase test, growth on mannitol salt agar &amp; Dnase agar). Using microgen staph identification kit, the isolates were further characterized into various Staph spp. Disc agar diffusion method was used for eleven (11) antibiotics susceptibility test while vancomycin screening agar and BMD (Broth Micro dilution) MIC test was used for vancomycin susceptibility evaluation. Indirect observation of biofilm was performed using congo red agar (CRA) and Microtitre plate assay method. PCR and sequencing were conducted on the isolates to molecularly detect the presence of 16SrRNA, IcaA, IcaB, IcaD genes.  Susceptibility of S aureus (21%) isolates tested against 12 different categories of antibiotics shows 4(27%) not multidrug resistant (MDR), 7(47%) were MDR and 4 (27%) were extensively drug resistant (XDR). High percentage (74%) of Multiple antibiotic resistant index at ≥0.3 suggested that the isolates originated from an environment where antibiotics are often used. Biofilm assessment of MDR S aureus isolates revealed that 9(82%) and 11 (100%) of the isolates were biofilm formers using Congo Red Agar method and Microtiter plate assay respectively. All isolates amplified with 16SrRNA primer signifying all are S. aureus, 100% of the isolates amplified with IcaA, 90% isolates amplified with IcaD, and 50% isolates amplified with IcaB. We can conclude that there is high prevalence of Biofilm forming S. aureus strains in the hospitals studied. This result should be born in mind by clinicians while prescribing medication for S. aureus positive conditions. Hospital managements should be proactive in preventing the preservation of the biofilm formers in their hospital environment through adequate sanitary and disinfection protocols. It is also necessary for clinician to note that nitrofurantoin, linezolid and vancomycin were most effective against the biofilm producing S. aureus and microtitre plate assay method was observed to be most reliable assay in the study of biofilm.

Detection of Antibiotic Resistance and Biofilm-Producing Ability of Staphylococcus Species in Clinical Isolates

Abstract Background Staphylococci are responsible for life-threatening infections in hospitals and community. Their ability to produce multiple virulence factors and antibiotic resistance is an important reason of high mortality in staphylococcal infections. Biofilm production by these organisms makes it difficult to treat. Most of the treating antibiotics are failing and making it a matter of concern. Aims This study aims to detect the increased antibiotic resistance in biofilm-producing Staphylococcus and to compare the performance of three potential methods of detection. Methods A total of 81 isolates of staphylococci including coagulase negative staphylococci (CoNs), methicillin resistant S. aureus (MRSA), and methicillin sensitive S. aureus (MSSA) are included in this study. After the identification, an antibiotic sensitivity test was performed. Biofilm detection was done by three different methods: Congo red agar method, tube adherence method, and microtiter plate method. Result Out of the 81 samples, 37 CoNs, 17 MRSA, and 27 MSSA were identified. Out of them we got 43 (53%) biofilm producers by Congo red agar method, 40 (49%) by tube adherence method, and 52 (64%) producers by tissue culture plate/microtiter plate method. Most of the biofilm producers showed multiple drug resistance. Conclusion We found out that the microtiter plate method is sensitive and reliable as compared with the other two methods. Antibiotic resistance was found to be very common in biofilm producers. This was due to the resistance developed as a result of the matrix that does not let the antibiotic bind with the organisms. This can make the treatment of Staphylococcus very difficult in the future as the rate of drug resistance is faster as compared with newly emerging antibiotics.

Biofilm formation and antibiotic resistance profiles of water-borne pathogens

Water sources (surface water, drinking water, rivers, and ponds) are significant reservoirs for transmitting antibiotic-resistant bacteria. In addition, these waters are an important public health problem because they are suitable environments for transferring antibiotic resistance genes between bacterial species. Our study aimed to assess the prevalence of Extended-spectrum beta-lactamase (ESBL) producing isolates in water samples, the susceptibility of the isolates to the specified antibiotics, the determination of biofilm ability, antibiotic resistance genes, and the molecular typing of the isolates. For this purpose, Polymerase chain reaction (PCR) and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses were used. Out of 70 isolates, 15 (21%) were ESBL producing, and sent for the MALDI-TOF analysis, where Escherichia coli, Acinetobacter calcoaceticus, Enterobacter bugandensis, Acinetobacter pittii, Pseudomonas aeruginosa, Acinetobacter junii, Pseudomonas oleovorans, and Enterobacter ludwigigii were identified. Moreover, colistin resistance genes (mcr 1/2/6, mcr 4, mcr 5, mcr 3/7, and mcr 8), ESBL-encoding genes (blaSHV, blaTEM, and blaCTX-M) and carbapenemase genes (blaNDM, blaOXA-48, and blaKPC) using molecular analysis (PCR) were confirmed. The colistin resistance gene was detected at 80% (12/15) in the isolates obtained. The distribution of these isolates according to resistance genes was found as mcr 1/2/6 4 (20%), mcr 3/7 3 (13%), and mcr 5 (40%). Additionally, the isolates harbored blaSHV(6.6%) and blaTEM (6.6%) genes. However, blaNDM, blaOXA-48, blaKPC, and blaCTX-M genes were not detected in any isolates. According to the Congo red agar method, seven (46.6%) isolates showed negative biofilm ability, and eight (53.3%) showed moderate biofilm ability. However, the microplate method detected weak biofilm in 53.3% of the isolates. In conclusion, this study provides evidence for the existence of multidrug-resistant bacteria that co-exist with mcr and ESBL genes in water sources. These bacteria can migrate to other environments and pose increasing threats to public health.