icaA Genes Research Articles

English

The current study aimed to investigate the prevalence and molecular characterization of biofilm-positive S. aureus isolates from bovine subclinical mastitis. The study also highlights the role of commonly used NSAIDs and ivermectin to modulate the S. aureus-associated antibiotic resistance. The results found a 41.41% S. aureus prevalence, out of which 25.79% isolates were biofilm-positive based on Congo red agar, microtitre plate test, and presence of icaA gene. Phylogenetic analysis of study isolates showed a high similarity with Egyptian and Indian icaA-positive S. aureus isolates. The comparative antibiotic resistance profiling showed a significantly (p<0.05) higher resistance to gentamicin, oxytetracycline, and cotrimoxazole by biofilm-positive isolates compared to non-biofilm forming isolates. The prevalence of methicillin and vancomycin resistant S. aureus was 62.5 and 20.83%, respectively. Antimicrobial effects of non-antibiotics against study isolates accessed through well diffusion method showed higher zones of inhibition for meloxicam followed by flunixin, ketoprofen, and ivermectin. The combinations of resistant antibiotics with non-antibiotics were investigated using well diffusion method and checkerboard assay. The combinations of amoxicillin/meloxicam, cotrimoxazole/flunixin, cotrimoxazole/ ketoprofen, and gentamicin/flunixin on well diffusion method and cotrimoxazole/ flunixin, amoxicillin/ketoprofen and gentamicin/flunixin on checkerboard assay revealed synergistic interactions. The study concluded that biofilm positive S. aureus is an emerging and prevailing cause of bovine mastitis in dairy farms of Pakistan. The increasing antibiotic resistance in S. aureus can be modulated by combining the resistant antibiotics with NSAIDs, especially flunixin and ketoprofen

Anti-biofilm effects of sinomenine against Staphylococcus aureus.

Staphylococcus aureus is a gram-positive foodborne pathogen capable of forming strong biofilms. This study identified that anti-biofilm natural compound against S. aureus. Sinomenine, a natural compound, showed significantly reduced biofilm formation (31.97-39.86%), but no effect on bacterial growth was observed. The dispersion of preformed biofilms was observed by confocal laser scanning microscopy (CLSM). qRT-PCR revealed that sinomenine treatment significantly up-regulated agrA by 3.8-fold and down-regulated icaA gene by 3.1-fold. These indicate that sinomenine treatment induces biofilm dispersal due to cell-cell adhesion, polysaccharide intercellular adhesin (PIA), and phenol-soluble modulin (PSM) peptides production. Our results suggest that sinomenine can be used as a promising agent for effectively controlling biofilm formation and dispersion, thereby making S. aureus more susceptible to the action of antimicrobial agents.

Effects of Thymus vulgaris and Cinnamomum verum Essential Oils on bap and ica Gene Expression in Staphylococcus aureus

Background: Staphylococcus aureus can cause a variety of infectious diseases, mainly due to its ability to shift between the planktonic and the biofilm lifestyle. Biofilm infections present a serious problem in human medicine. Biofilm-associated protein (bap) and intercellular adhesin (ica) genes are involved in biofilm formation. Objectives: This study evaluated the effect of Thymus vulgaris and Cinnamomum verum essential oils on biofilm formation and expression of icaA, icaD, and bap genes in S. aureus strains. Methods: A total of 20 strains of S. aureus were isolated from the urine of patients. The susceptibility test was performed to determine the effect of minimum inhibitory concentration (MIC) of thyme and cinnamon essential oils on strains by broth dilution method. The expression of icaA and icaD genes was determined by measuring the cognate messenger ribonucleic acid level using real-time polymerase chain reaction (PCR). Results: The biofilm formation of strains after treatment with the MIC of thyme essential oil was observed as 40% weak and 60% negative biofilms, and with the MIC of cinnamon essential oil as 25% strong and 75% moderate biofilms. Real-time PCR demonstrated that 60% of S. aureus strains treated with thyme and cinnamon essential oils showed a decrease in icaA gene expression. Moreover, 55% and 70% of the strains treated with thyme and cinnamon essential oils had reduced icaD gene expression, respectively. None of the strains of S. aureus had a bap gene. Conclusions: The present study showed that thyme and cinnamon essential oils reduce the expression of icaA and icaD genes, and cinnamon essential oil is more effective than thyme essential oil.

Effect of MA01 rhamnolipid on cell viability and expression of quorum-sensing (QS) genes involved in biofilm formation by methicillin-resistant Staphylococcus aureus

A group of biosurfactants, called rhamnolipids, have been shown to have antibacterial and antibiofilm activity against multidrug-resistant bacteria. Here, we examined the effect of rhamnolipid biosurfactants extracted from Pseudomonas aeruginosa MA01 on cell growth/viability, biofilm formation, and membrane permeability of methicillin-resistant Staphylococcus aureus (MRSA) ATCC6538 bacterial cells. The results obtained from flow cytometry analysis showed that by increasing the concentration of rhamnolipid from 30 to 120 mg/mL, the cell viability decreased by about 70%, and the cell membrane permeability increased by approximately 20%. In fact, increasing rhamnolipid concentration was directly related to cell membrane permeability and inversely related to cell survival. Microtiter plate biofilm assay and laser scanning confocal microscopy analysis revealed that rhamnolipid, at a concentration of 60 mg/mL, exerts a reducing effect on the biofilm formation of Staphylococcus aureus. Real-time PCR analysis for monitoring the relative changes in the expression of agrA, agrC, icaA, and icaD genes involved in biofilm formation and related to the quorum-sensing pathway after treatment with rhamnolipid indicated a reduced expression level of these genes, as well as sortase A gene. The results of the present study deepen our knowledge regarding the use of microbial natural products as promising candidates for therapeutic applications.

Antibiotic resistance, biofilm formation, and virulence factors of isolates of staphylococcus pseudintermedius from healthy dogs and dogs with keratitis.

Canine bacterial keratitis is a common infection that can potentially threaten vision. Staphylococcus pseudintermedius (S. pseudintermedius) is an opportunistic pathogen that has been isolated from the canine conjunctival sac but there are only a few reports on the role of this bacterium in canine keratitis. This study focused on the distribution rate of S. pseudintermedius in the canine conjunctival sac, and the antibiotic resistance, biofilm-producing ability, and dissemination of virulence factors in strains of S. pseudintermedius isolated from healthy dogs and dogs with keratitis. The study included 35 healthy dogs and 40 dogs with keratitis. Bacterial species were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI–TOF MS). Strains of S. pseudintermedius were screened for resistance against nine different antibiotics by the Kirby–Bauer assay. The ability to produce biofilm was investigated by microtiter plate assay (MtP) and amplification of icaA and icaD genes. Virulence factors in the strains were also evaluated. A total of 132 aerobic bacteria were isolated from the 119 samples in the study. Among them, 67 bacterial strains were isolated from 70 eyes of healthy dogs, and 65 bacterial strains were isolated from 49 eyes of dogs with keratitis. The prevalence of S. pseudintermedius, which was the most frequent bacterial isolate in both the groups, was 20.9% in the healthy group and 23.08% in the keratitis group. Most of the isolates of S. pseudintermedius were sensitive to rifampin (96.6%), oxacillin (100%), and neomycin (96.6%), and resistant to tetracycline (96.6%). Virulence factors such as lip (96.6%), hlgB (96.6%), and hlgA (96.6%) were found in most of the isolates, and 89.66% of isolates were classed as biofilm producers. In conclusion, S. pseudintermedius was the common bacterium in the conjunctivital sac of the healthy dogs and dogs with keratitis in Yangzhou, China, and the presence of virulence factors and biofilm-formation ability were high in the strains isolated from the dogs with keratitis.

Analysis of antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery.

To analyze antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery with the focus on coagulase-negative staphylococci (CNS) during cataract surgery and discuss the implications of this colonization as a potential risk of acquiring endophthalmitis. After approval of the institutional review board and informed consent from patients had been obtained, conjunctival swabs for culture from 59 patients undergoing cataract surgery were taken of the fellow eye at baseline (C0) and from the eye to be operated before (T0) and after (T1) irrigation with povine-iodine 5%, and at the end of surgery (T2). Genes responsible for virulence (mecA, ica and atlE) and antibiotic profile were determined; strain clonality of persistent colonizing Staphylococcus epidermidis strains was established by the Multi-locus sequence typing (MLST). The frequency of CNS was significantly reduced in T1 (13.6%) from 81.4% in T0 and 86.4% in C0. The frequency of mecA, ica and atlE genes was 34.4%, 37.5% and 61.4%, respectively; and methicillin phenotypic resistance was 35.4%. S. epidermidis was the most frequent species isolated in every time point. MLST revealed in 7 patients 100% coincidence of the seven alleles of the S. epidermidis isolated previous to povine-iodine 5% disinfection and at the end of the surgery. CNS isolates from T1 or T2 corresponded to the same species, antibiotic and virulence profile as those isolates from C0 or T0. Povidone-iodine 5% prophylaxis before surgery significantly reduced conjunctival contamination; in those that persisted, the source of contamination was mostly the patient's microbiota confirmed by the MLST system.

Investigation of Biofilm Formation Properties of Coagulase Negative Staphylococci Isolated from Catheter-Related Bloodstream Infections

In view of the significant negative impact of biofilm-mediated infection on patient health and the necessity of a reliable phenotypic method to detect biofilm producers, this study aimed to demonstrate phenotypic and molecular biofilm formation in coagulase-negative staphylococci (CoNS) isolated from catheter related infections and to compare the methods used with each other. The study was also aimed to determine the biofilm eradication effect of vancomycin in order to guide for the treatment. For the detection of biofilm formation, a total of 154 CoNS clinical isolates of which 30 being causative agents of catheter related bloodstream infection (CRBSI) (isolated from both the catheter tip and blood cultures of 15 patients), 89 being isolated from peripheral blood cultures of patients without a central venous catheter (CVC) (13 of them were bloodstream infection agents, 76 of them were contaminant), and 35 being isolated as catheter colonizer, were screened by tissue culture plate (TCP), Congo red agar (CRA) method and polymerase chain reaction (icaA, icaD and IS256). Vancomycin minimum inhibitory concentration (MIC) and minimum biofilm eradicating concentration (MBEC) values were determined. The pulsed field gel electrophoresis (PFGE) method was used to show the clonal relationship between CoNS isolated from the catheter tips and peripheral blood of patients with CRBSI. Of the 154 CoNS isolates included in the study, 38.9% were Staphylococcus epidermidis (n= 60), 34.4% were Staphylococcus haemolyticus (n= 53), 20.7% were Staphylococcus hominis (n= 32), and 3.8% were detected as Staphylococcus capitis (n= 6). In our study, biofilm formation was shown in 31.8% with the CRA method and in 68.1% with the TCP method. By TCP method, 22% (n= 34) were determined as weak, 31.2% (n= 48) medium and 14.9% (n= 23) strong biofilm producers. While the sensitivity of the CRA method was found to be low for isolates that were determined as weak positive in the microplate method, the high sensitivity of the CRA method for isolates with medium and strong positivity was found remarkable. The positivity rates of icaA, icaD and IS256 genes in a total of 154 CoNS isolates were found to be 40 (25.9%), 57 (37%) and 77 (50%), respectively. In total, at least one gene positivity was detected in 107 (69.5%) isolates. Single gene positivity was detected in 55 (35.7%), two gene positivity in 35 (22.7%) and three gene positivity was detected in 17 (11%) of the included CoNS. Biofilm formation (four weak, four medium, two strong) was detected by microplate method in 10 of 47 CoNS isolates (five S.epidermidis, three S.hominis, one S.haemolyticus and one S.capitis) in which no genes were detected. Vancomycin MBEC/ MIC values were found to be high and it was observed that as the biofilm forming power of the isolates increased, the MBEC/MIC ratio also increased. The CoNS isolated from the catheter samples and blood of patients diagnosed with CRBSI had a 100% similar profile with PFGE except for one unevaluable isolate. The tissue culture plate (TCP) method was found to be most sensitive, accurate and reproducible screening method for detection of biofilm formation by staphylococci and has the advantage of being a quantitative model to study the adherence of staphylococci. The presence of the icaAD and IS256 gene is not always associated with in vitro biofilm formation. For this reason, it is more appropriate to use more than one method together for the evaluation of biofilm formation. It was thought that the use of reliable methods to specifically detect biofilms could be helpful in diseases that are difficult to treat. Considering the high rates of biofilm and antimicrobial resistance of biofilm-forming isolates in biomedical device associated infections, it was determined that it would not be sufficient to evaluate only the MIC results for susceptibility results.

Persistence of Staphylococcus spp. in milk from cows undergoing homeopathy to control subclinical mastitis

BackgroundMastitis is one of the major diseases in dairy cattle, as it causes great economic losses to producers due to the reduction of milk production and changes in the quality of the product. The disease is mainly caused by bacteria of the genus Staphylococcus spp., these microorganisms can express various virulence factors, such as biofilms for example. In herds with organic management, producers and technicians use unconventional ways to treat and control the disease, such as homeopathy. However, it is not known if this type of treatment is able to control pathogenic bacteria such as those of the genus Staphylococcus, of relevance to animal and human health. Thus, the objective of this study was to investigate the production of biofilm in vitro and its genes by Staphylococcus spp. isolated in the milk of cows treated with homeopathy, as well as the persistence of microorganisms in animals.MethodsNinety-nine isolates of Staphylococcus spp. from cows treated and not treated with homeopathy were identified by internal transcribed space-polymerase chain reaction and investigated for the presence of the icaABCD, bap, aap, atlE, and bhp genes and in vitro biofilm production using the adhesion method on polystyrene plates. The enzyme restriction profile was determined by Pulsed-Field Gel Electrophoresis. Clusters of S. aureus and S. epidermidis with three or more isolates had an isolate selected for Multilocus Sequence Typing.ResultsThe frequency of S. aureus isolations was similar in treated and untreated cows, while 71.4% of the coagulase-negative identified were isolated in cows treated with homeopathy. The distribution of the operon ica genes was similar in animals with and without treatment, except for the icaD gene, more frequent in treated cows. Production of biofilm was associated with presence of one or more genes from the icaADBC operon. S. aureus revealed a greater diversity and greater dissemination in cows treated and not treated with homeopathy. Sequence Types ST1, ST5, and ST126 were identified in S. aureus.ConclusionsThe presence of biofilm-associated genes and the in vitro production of biofilms, combined with the persistence of clonal profiles of Staphylococcus spp. demonstrate other forms of control for bovine mastitis should be researched for organic production herds.

Phenotypic and Genotypic Detection of Biofilm-Forming Staphylococcus aureus from Different Food Sources in Bangladesh.

Simple SummaryBiofilm formation by Staphylococcus aureus in foods poses a potential concern for public health and food safety. Therefore, the present study was conducted to detect biofilm-producing S. aureus from foods and human hand swabs using phenotypic and genotypic assays. In this study, S. aureus was detected in 23.81% (100/420) of samples, and among them, 89 and 97 of the isolates were biofilm producers by qualitative and quantitative tests, respectively. At least one biofilm-forming gene was detected in 21 S. aureus isolates, of which four isolates harbored all five adhesion genes (icaA, icaB, icaC, icaD, and bap). In addition, the occurrence of adhesion genes in S. aureus isolates showed a strong significant correlation among themselves. This is the first report on detecting biofilm-forming S. aureus from foods and hand swabs in Bangladesh using the molecular technique. The findings from this study indicate a significant public health risk and suggest the necessity of maintaining food hygiene practices at every step of the food chain to prevent and control S. aureus foodborne illness.Staphylococcus aureus is a major foodborne pathogen. The ability of S. aureus to produce biofilm is a significant virulence factor, triggering its persistence in hostile environments. In this study, we screened a total of 420 different food samples and human hand swabs to detect S. aureus and to determine their biofilm formation ability. Samples analyzed were meat, milk, eggs, fish, fast foods, and hand swabs. S. aureus were detected by culturing, staining, biochemical, and PCR. Biofilm formation ability was determined by Congo Red Agar (CRA) plate and Crystal Violet Microtiter Plate (CVMP) tests. The icaA, icaB, icaC, icaD, and bap genes involved in the synthesis of biofilm-forming intracellular adhesion compounds were detected by PCR. About 23.81% (100/420; 95% CI: 14.17–29.98%) of the samples harbored S. aureus, as revealed by detection of the nuc gene. The CRA plate test revealed 20% of S. aureus isolates as strong biofilm producers and 69% and 11% as intermediate and non-biofilm producers, respectively. By the CVMP staining method, 20%, 77%, and 3% of the isolates were found to be strong, intermediate, and non-biofilm producers. Furthermore, 21% of S. aureus isolates carried at least one biofilm-forming gene, where icaA, icaB, icaC, icaD, and bap genes were detected in 15%, 20%, 7%, 20%, and 10% of the S. aureus isolates, respectively. Bivariate analysis showed highly significant correlations (p < 0.001) between any of the two adhesion genes of S. aureus isolates. To the best of our knowledge, this is the first study in Bangladesh describing the detection of biofilm-forming S. aureus from foods and hand swabs using molecular-based evidence. Our findings suggest that food samples should be deemed a potential reservoir of biofilm-forming S. aureus, which indicates a potential public health significance.

Phenotypic and Molecular Detection of Slime Producing Staphylococcus Spp. Obtained from Blood Samples of Patients Undergoing Hematopoietic Stem-Cell Transplantation

Abstract Aim: to investigate the slime production in isolates of Staphylococcus spp., associated with bacteremia in patients after hematopoietic stem-cell transplantation (HSCT) and to determine the relationship between the slime production and ica genes carriage, as well as the correlation of ica and methicillin resistance. Materials and methods: Between 2019 and 2020, twenty-one clinically significant Staphylococcus spp. isolates were obtained from blood cultures of 17 patients after HSCT. The species identification and the susceptibility to cefoxitin were determined by BD Phoenix M50. Two phenotypic tests (Congo red agar, CRA; Christensen’s method, TT) and PCR for icaA and icaD were used to detect slime production. A PCR method was also used to detect the mecA, mecC genes. Results: In the studied group of 21 isolates (S. epidermidis, n = 12; S. haemolyticus, n = 4; S. hominis, n = 2; S. aureus, n = 3), the phenotypic tests were positive in 13 isolates. Ten isolates (47.6%) were identified as carriers of ica genes (S. epidermidis, n = 9, and S. haemolyticus, n = 1). Five isolates (23.8%) were detected as slime producers by all three methods. The mecA gene was identified in 18 isolates (85.7%). All ica positive isolates were also mecA carriers. Conclusion: A relatively high proportion of the blood isolates of Staphylococcus spp. were slime producers, associ-ated with ica genes. A combination of both phenotypic and genetic methods should be used to detect alternative routes of slime production. The co-expression of ica and mecA is associ-ated with the occurrence of difficult-to-eradicate isolates.

Susceptibility to Vancomycin of Biofilm Producing Staphylococci Isolated from Tertiary Care Hospital of Nepal

BACKGROUND: Methicillin resistance and biofilm-producing Staphylococci are emerging as multidrug-resistant strains narrowing the efficacy of antimicrobial therapy. Although vancomycin is used as the drug of choice to treat such isolates, different studies worldwide have documented the emergence of strains that are intermediately susceptible or resistant to this antibiotic. OBJECTIVE: The study aimed to determine the minimum inhibitory concentration of vancomycin to methicillin-resistant and biofilm-producing staphylococci isolated from different clinical specimens. METHODS: 375 staphylococci isolated from different clinical specimens over one year were included in the study. Biofilm formation was determined by the Tissue culture plate method (TCP), and ica genes were identified by Polymerase Chain Reaction (PCR). Antibiotic susceptibility and methicillin resistance were done following Clinical and Laboratory Standards Institute (CLSI) guidelines. The minimum inhibitory concentration (MIC) of vancomycin in all isolates was determined by the agar dilution method. RESULTS: Among 375 Staphylococci studied, 43% and 57% represented S. aureus and Coagulase-Negative Staphylococci (CNS), respectively. The rate of Methicillin-Resistant S. aureus (MRSA) and Methicillin-Resistant Coagulase Negative Staphylococci (MRCNS) were 81.4% and 66.8% respectively and determined by the disc diffusion method. The most potential antibiotics were tetracycline and chloramphenicol showing sensitivity to more than 90% isolates. The Minimum Inhibitory Concentration (MIC) value of oxacillin for staphylococci ranged from 0.125-32 μg/ml. Oxacillin agar diffusion method showed 51.6% and 79.9% isolates as MRSA and MRCNS, respectively, revealing a very high percentage of S. aureus and CNS isolates as methicillin-resistant. All isolates had susceptible vancomycin MICs that ranged from 0.125-2 μg/ml. Two S. aureus isolated from Central Venous Catheter (CVC) and catheter specimens were detected with intermediate susceptibility to vancomycin. Similarly, three CNS isolated from blood, CVC, and wound/pus (w/p) were intermediately susceptible to vancomycin. Strong biofilm formation was observed in 22.1% of clinical isolates, and the ica gene was detected among 22.9% of isolates. Only one S. aureus detected as a biofilm producer by the TCP method was found to have intermediate susceptibility to vancomycin. CONCLUSIONS: The increment in vancomycin MIC among methicillin-resistant and biofilm-producing staphylococci is alarming. Strict control measures to prevent methicillin-resistant isolates spread and routine surveillance for vancomycin-resistant isolates must be incorporated in hospitals to prevent antimicrobial treatment failure.

Delivery LL37 by chitosan nanoparticles for enhanced antibacterial and antibiofilm efficacy

In this study, the fabrication of LL37-loaded chitosan nanoparticles (CS/LL37-NPs) was based on an ionotropic gelation method between sodium tripolyphosphate (TPP) and chitosan. Synthesized chitosan nanoparticles (CS-NPs) were approved by Fourier Transform Infrared (FTIR), UV–vis spectroscopy, Dynamic Light Scattering (DLS), Scanning Electron Microscope (SEM), and Transmission Electron Microscopy (TEM). The encapsulation efficiency of LL37 in this delivery system (CS/LL37-NPs) was 86.9%. According to in vitro release profile, the release of LL37 from CS/LL37-NPs was almost complete after 5 days. Additionally, CS/LL37-NPs can cause an increase in the half-life and prolonged LL37 antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA). This delivery system demonstrated 68% biofilm formation inhibition compared to the LL37 alone. Also, icaA gene expression in the face of CS/LL37-NPs was significantly decreased. This study showed the important role of delivery systems in enhancing LL37 antibacterial and antibiofilm activity which can be suggested as a promising agent in the inhibition of bacterial growth and the prevention of biofilm formation.

Linezolid decreases Staphylococcus aureus biofilm formation by affecting the IcaA and IcaB proteins.

The ica gene of Staphylococcus aureus (S. aureus) plays a vital role in its growth and biofilm formation. Among them, IcaA and IcaB are critical proteins for synthesizing extracellular polysaccharides and biofilms in S. aureus. To investigate whether the formation of S. aureus biofilms can be inhibited through the IcaA and IcaB proteins by the presence of linezolid. The icaA and icaB genes of S. aureus ATCC 25923 were silenced by homologous recombination. The critical roles of icaA and icaB in S. aureus were analysed by observing the growth curve and biofilm formation after linezolid treatment. Then, the effect of linezolid on the morphology of S. aureus was observed by scanning electron microscopy. Finally, the potential binding ability of linezolid to Ica proteins was predicted by molecular docking. The icaA- and icaB-silenced strains were successfully constructed, and the sensitivity of S. aureus to linezolid was decreased after icaA and icaB silencing. Scanning electron microscopy showed that linezolid caused invagination of the S. aureus surface and reduced the production of biofilms. Molecular docking results showed that linezolid could bind to IcaA and IcaB proteins. IcaA and IcaB are potential targets of linezolid in inhibiting the biofilm formation of S. aureus (ATCC 25923).

Expression of icaA, B, D, R and ebps biofilm-associated genes in methicillin-resistant-Staphylococcus aureus in exposure to curcumin

BackgroundBiofilm formation is a paramount event to develop withstanding and sustainable biofilms by bacterial species. Methicillin-resistant Staphylococcus aureus (S. aureus, MRSA) causes a variety of infections. Our aim was evaluation of effect of curcumin compound on the expression of icaA, B, D, R and ebps genes related to biofilm formation among MRSA from blood samples. MethodsOne-hundred MRSA were collected from blood samples. Various concentrations of curcumin were assessed for antimicrobial effect including 1, 2, 5, 10 and 20 mg/mL in 96-well plates and incubated for 24 h. Curcumin at concentration of 2 mg/mL was prepared and exposed to bacterial culture (with a suspension equal to 0.5 Mac Farland) and incubated for 24 h. The expression of icaA, B, R and ebps genes related to biofilm formation was measured in exposure to 2 mg/mL of curcumin using real-time PCR method. ResultsPrevious hospitalization (58%, p = 0.0372) and antibiotic consumption (76%, p = 0.0023) were found as major risk factors of MRSA isolation. The minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) of curcumin ranged 5–10 and 10- > 20 mg/mL, respectively. The curcumin exerted a significantly higher anti-biofilm effect at concentrations 5, 10 and 20 mg/mL compared to the control group. The expression of icaA, B, D and ebps genes decreased 1.8 fold (p = 0.011), 2 fold (p = 0.002), 2.3 fold (p < 0.001) and 3 fold (p < 0.001), respectively which was significant, but the expression of the icaR was not significantly decreased (0.3 fold, p = 0.339). ConclusionThe curcumin compound had antimicrobial effect against MRSA. The expression of biofilm-associated genes was decreased in exposure to sub-MIC concentration of curcumin. Therefore, curcumin has the potential of application as an alternative antimicrobial agent.

Identifying biofilm-forming strains of Staphylococcus epidermidis isolated from intravascular-catheterized patients by icaA and icaD genes

Abstract Staphylococcus epidermidis has gained a substantial importance in recent years because it is one of the main causative agents of nosocomial infections. It requires a predisposed host in order to switch from a normal inhabitant of the human skin to a pathogenic flora. This study aimed to use icaA/icaD genes as biomarkers in differentiating biofilm-forming S. epidermidis, obtained from patients with intravascular catheter (IVC) infections, from other saprophytic strains. Twenty isolates of S. epidermidis obtained from 100 cases of intravascular catheter infections were investigated for the presence of the intracellular adhesion icaA and icaD genes by polymerase chain reaction (PCR) and for phenotypic biofilm production by qualitative Congo red agar assay (CRA). Results: Nine (~45%) S. epidermidis isolates out of 20 isolates collected from IVC infections were positive for both CRA (produce black colonies) and icaA/icaD genes; while 11 (~55%) S. epidermidis isolates were negative for CRA and icaA/icaD genes. Detection of icaA/icaD gene is a reliable, efficient, and more rapid tool for characterizing biofilm-forming strains of S. epidermidis. Keywords: Staphylococcus epidermidis; icaA; icaD; biofilm

Evaluation of the Pathogenic-Mixed Biofilm Formation of Pseudomonas aeruginosa/Staphylococcus aureus and Treatment with Limonene on Three Different Materials by a Dynamic Model.

Background: Biofilms have been found growing on implantable medical devices. This can lead to persistent clinical infections. The highly antibiotic-resistant property of biofilms necessitates the search for both potent antimicrobial agents and novel antibiofilm strategies. Natural product-based anti-biofilm agents were found to be as efficient as chemically synthesized counterparts with fewer side effects. In the present study, the effects of limonene as an antibiofilm agent were evaluated on Pseudomonas aeruginosa and Staphylococcus aureus biofilm formed on different surfaces using the CDC model system in continuous flow. The flgK gene and the pilA gene expression in P. aeruginosa, and the icaA gene and eno gene in S. aureus, which could be considered as efficient resistance markers, were studied. Methods: Mono- and dual-species biofilms were grown on polycarbonate, polypropylene, and stainless-steel coupons in a CDC biofilm reactor (Biosurface Technologies, Bozeman, MT, USA). To evaluate the ability of limonene to inhibit and eradicate biofilm, a sub-MIC concentration (10 mL/L) was tested. The gene expression of P. aeruginosa and S. aureus was detected by SYBR Green quantitative Real-Time PCR assay (Meridiana Bioline, Brisbane, Australia). Results: The limonene added during the formation of biofilms at sub-MIC concentrations works very well in inhibiting biofilms on all three materials, reducing their growth by about 2 logs. Of the same order of magnitude is the ability of limonene to eradicate both mono- and polymicrobial mature biofilms on all three materials. Greater efficacy was observed in the polymicrobial biofilm on steel coupons. The expression of some genes related to the virulence of the two microorganisms was differently detected in mono- and polymicrobial biofilm. Conclusions: These data showed that the limonene treatment expressed different levels of biofilm-forming genes, especially when both types of strains alone and together grew on different surfaces. Our findings showed that limonene treatment is also very efficient when biofilm has been grown under shear stress causing significant and irreversible damage to the biofilm structure. The effectiveness of the sanitation procedures can be optimized by applying antimicrobial combinations with natural compounds (e.g., limonene).

The Association between icaA and icaB Genes, Antibiotic Resistance and Biofilm Formation in Clinical Isolates of Staphylococci spp.

Sixty-six (66) Staphylococcus bacterial isolates were withdrawn from separate clinical samples of hospitalized patients with various clinical infections. Conventional bacteriological tests identified the species of all isolates, and standard microbiological techniques differentiated them into CoPS or CoNS. Their biofilm development was followed by an analysis via the MTP (microtiter tissue culture plates) technique, and we then investigated the presence/absence of icaA and icaB, which were qualified in the top-30 potent biofilm-forming isolates. Thirteen isolates (46.7%) showed the presence of one gene, six (20%) isolates exhibited the two genes, while ten (33.3%) had neither of them. The formation of staphylococci biofilms in the absence of ica genes may be related to the presence of other biofilm formation ica-independent mechanisms. CoPS was the most abundant species among the total population. S. aureus was the sole representative of CoPS, while S. epidermidis was the most abundant form of CoNS. Antibiotic resistance was developing against the most frequently used antimicrobial drugs, while vancomycin was the least-resisted drug. The totality of the strong and medium-strength film-forming isolates represented the majority proportion (80%) of the total investigated clinical samples. The biochemical pattern CoPS is associated with antibiotic resistance and biofilm formation and can be an alarming indicator of potential antibiotic resistance.

Elucidating the antibiofilm activity of Frangula emodin against Staphylococcus aureus biofilms.

Because the Staphylococcus aureus is one of the most well-known pathogens associated with medical devices and nosocomial infections, the aim of the study was to examine antibiofilm potential of emodin against it. Antibacterial activity was examined through microdilution assay. Antibiofilm testing included crystal violet staining of biofilm biomass and morphology analysis by Atomic force microscopy (AFM). Furthermore, aerobic respiration was monitored using the Micro-Oxymax respirometer. For investigation of gene expression qRT-PCR was performed. Emodin demonstrated strong antibacterial activity and ability to inhibit biofilm formation of all tested strains. The effect on preformed biofilms was spotted in few strains. AFM revealed that emodin affects biofilm structure and roughness. Monitoring of respiration under emodin treatment in planktonic and biofilm form revealed that emodin influenced aerobic respiration. Moreover, qRT-PCR showed that emodin modulates expression of icaA, icaD, srrA and srrB genes, as well as RNAIII, and that this activity was strain-specific. The results obtained in this study indicate the novel antibiofilm activity of emodin and its multiple pathways of action. This is the first study that examined pathways through which emodin expressed its antibiofilm activity.

Biofilm Formation and Antibiotic Resistance of S. aureus Strains isolated from Chronic Traumatic Wounds

Staphylococcal biofilms are the prominent cause of chronic wound infection and antibiotic resistance. It acts as a reservoir for bacteria, making wound healing difficult. Biofilm infections increase the hospital stays and cost to the patients. The current study explores the phenotypic and genotypic detection of S. aureus biofilm from chronic traumatic wounds and their association with antibiotic resistance. A prospective observational study was conducted from April 2020 to March 2021. S. aureus isolates were identified by the MALDI-TOF. Antibiotic susceptibility was determined by VITEK 2. Biofilm production detected by tissue culture plate method and associated ica genes were diagnosed through multiplex PCR. Overall, 67 isolates were investigated. The frequency of biofilm production in S. aureus was 73.1%, and most of the isolates were moderate biofilm producers (38.8%). The presence of intracellular adhesion (ica) operon among these isolates was 85.7% and also significantly associated with the strength of biofilm mass formation. Ica A was the predominant biofilm-producing gene (42.9%). Biofilm producing Methicillin-resistant S. aureus were as high as 75%, and multidrug resistant strains were significantly associated with biofilm formation. But frequency of ica genes were noted more in Methicillin sensitive S. aureus (65.2%). High frequency of biofilm in S. aureus of isolates was responsible for the development of chronic non-healing traumatic wounds. Biofilm-producing isolates showed greater multidrug-resistance. Phenotypically MRSA expressed more biofilm, but ica operon was documented in MSSA. It emphasized the further need for ica independent pathway exploration for MRSA biofilm synthesis.

DETECTION OF INTRACELLULAR ADHESION GENE (icaA and icaD)AND BIOFILM FORMATION STAPHYLOCOCCUS AUREUS ISOLATES FROM MASTITIS MILK OF SHEEP AND GOAT

In the present study, a total of 150 mastitis milk samples were collected from sheep andgoat (75 for each one) and were analyzed for the presence of S.aureus.. The obtainedresults indicate that this bacterium observed in 20% of these samples (21.33% from sheepand 18.66% from goat) .The study of antibiotic susceptibility test to 9 different antibioticsshowed that S. aureus was 100% resistant to penicillin and 100% sensitive tovancomycin, gentamycin , clarithromycin and chloramphenicol . whereas for cefoxitin(alternative to methicillin) resistance was 47%.There were a variable sensitivitypercentage for the rest of antibiotics: Tetracycline (70%), Ciprofloxacin (80%) ,Clindamycin (83%). The biofilm-forming ability of S. aureus was evaluated viamicrotiter plates and the result revealed that, all the studied isolates were either moderatebiofilm producer or weak biofilm producer while the non-biofilm producer and strongbiofilm producer were not detected among the tested isolate.The relationship betweenbiofilm formation and resistance to methicillin showed there as no significant differences(P>0.05) in the percentage of weak and moderate biofilm producers between MRSA andMSSA isolates. PCR analysis was applied to DNA extracted from S.aureus isolates frommilk samples .The results of PCR assay revealed that all S.aureus isolates gave positiveresults for both icaA and icaD genes (100%) with Product size 151 and 211 bp,respectively.Results of this study indicate that biofilm producing S.aureus have a major role player onthe occurrence of mastitis .In addition, there was high prevalence of MRSA isolates(47%) in mastitic milk at the study area