icaD Genes Research Articles

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.

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.

Screening for Intercellular Adhesion Gene Cluster Genes Staphylococcus aureus Isolated from UTI Cases

144 urine samples were collected from persons suspected of urinary tract infections, their ages ranged from 7 to 60 years. Some pathogens of urinary tract infections were investigated, diagnosed, and antibiotic sensitivity tested. The percentage of patients with diabetes and pregnant women was also investigated, in addition to comparing patients between age and gender. The results showed the results of isolation and diagnosis of Staphylococcus aureus showed 15.27% of the number of (22) samples and were divided into age groups. The present study evaluated the ability of biofilm formation and the presence of (icaA) and (icaD) genes among strains of staphylococci isolated from patients with urinary tract infection. The presence of (icaA) and (icaD) gene was also determined by PCR technique the Biofilm development is common among Staphylococcus aureus isolated from urinary tract infections .The sensitivity of Staphylococcus aureus bacteria was tested to 10 antibiotics, and the highest sensitivity was to an antibiotic Methicillin 91%, and 86.3% towards Amoxicillin. It also gave an 81%percentage to an antibiotic Levofloxacin and Gentamicin. It is also being less sensitive to an antibiotic Ethromycin. The bacteria showed moderate sensitivity to an antibiotic Rifampicin 86.36% and to an antibiotic vancomycin 81.81% and it was less moderate sensitivity to an antibiotic Methicillin and Ethromycin 4.54%. The bacteria showed a high resistance to an antibiotic Ethromycin 90.90% and also to an antibiotic Tetracycline 81.81%, While there was no resistance to an antibiotic Rifampicin 0.0%. Through the study of the statistical analysis, it was shown that there are significant differences, that is, that there are differences in the effect of antibiotics on bacteria, and it was the best four antibiotics and had an effect on the sensitivity of bacteria it is Methicillin, Gentamicin, Levofloxacin and Ciprofloxacin.

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.

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

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.

Isolation of Biofilm Producing Bacteria from Stool Samples and Their Antibiogram

Aim: Bacterial biofilm formation is a menacing attribute that has been linked to a rise in antibiotic resistance. The aim of this study was to isolate biofilm-producing bacteria from stool samples and their antibiogram. Study Design: The study involved laboratory research, statistical analysis and interpretation of the data. Place and Duration of Study: The research was carried out in three (3) hospital facilities. They are University of Port Harcourt Teaching Hospital (UPTH), Meridian Hospital D/line branch (MRD1), and Meridian Hospital Ikoku branch (MDR2). Specimens were gathered within three (3) months, and analyses were performed. Methodology: 45 stool specimens were collected from the three (3) hospitals. The specimens were correctly labelled with the sampling date and time. Standard microbiological procedures were performed on the collected specimens, including plate counts, identification, biofilm screening, sensitivity testing, extended spectrum beta lactamase phenotypic screening, and molecular characterization of the isolates. Results: The total heterotrophic bacterial counts ranged from 6.2 to 8.2 x107 cfu/g, total coliform counts ranged from 3.2 to 4.1 x106 cfu/g and faecal coliform counts ranged from 1.3 to 1.4 x105 cfu/g. There was no significant difference at (p≤0.05) in the total heterotrophic bacterial counts, total and faecal coliform counts between the hospitals sampled. A total of Thirty-two (32) bacterial isolates were identified in stool specimens, with 20 (62.5%) of them being biofilm producers. Staphylococcus 30%, 35% Escherichia coli, 25% Enterococcus and 10% Bacillus species were detected among biofilm bacteria. Biofilm isolates showed a variety of susceptibility patterns and antibiotic resistance was found in biofilm bacteria. Most bacterial intestinal tract infections from patients and hospitals investigated can be treated with ofloxacin, Gentamycin, Imipenem, and Nitrofurantoin. TET A and CTX-M genes, were reported in Bacillus subtilis and Escherichia coli biofilm bacteria as possible genes that could confer antibiotic resistance.The existence of the icaD and papC genes in Bacillus subtilis and Escherichia coli has been discovered to be probable determinants that impart biofilm forming abilities, according to genomic studies. Conclusion: The existence of biofilm-producing bacteria in patients’ stools, as well as their antibiotic resistance, was observed in this study. Ceftazidime (third generation cephalosporin) resistance was found in both biofilm and non-biofilm bacteria. This research reveals that ofloxacin, Gentamycin, Imipenem, and Nitrofurantoin are the drugs of choice for bacterial intestinal tract infections caused by Escherichia coli, Staphylococcus, Enterococcus, and Bacillus species. As a result, proper infection control techniques and therapeutic recommendations for proven infections should be swiftly implemented.

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

Methicillin- and Vancomycin-Resistant Staphylococcus aureus From Humans and Ready-To-Eat Meat: Characterization of Antimicrobial Resistance and Biofilm Formation Ability.

Methicillin-resistant and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) are zoonotic life-threatening pathogens, and their presence in food raises a public health concern. Yet, scarce data are available regarding MRSA and VRSA in both ready-to-eat (RTE) meat and food handlers. This study was undertaken to determine the frequency, antimicrobial resistance, and biofilm-forming ability of MRSA and VRSA isolated from RTE meat (shawarma and burger) and humans (food handlers, and hospitalized patients) in Zagazig city, Sharkia Governorate, Egypt. We analyzed 176 samples (112 human samples: 72 from hospitalized patients and 40 from food handlers, 64 RTE meat samples: 38 from shawarma and 26 from burger). Using phenotypic, PCR-based identification of nuc gene and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 60 coagulase-positive S. aureus (COPS) isolates were identified in the samples as follow: RTE meat (15/64, 23.4%), hospitalized patients (33/72, 45.8%) and food handlers (12/40, 30%). All the COPS isolates were mecA positive (and thus were classified as MRSA) and multidrug resistant with multiple antibiotic resistance indices ranging from 0.25 to 0.92. Overall, resistance to cefepime (96.7%), penicillin (88.3%), were common, followed by ampicillin-sulbactam (65%), ciprofloxacin (55%), nitrofurontoin (51.7%), and gentamicin (43.3%). VRSA was detected in 30.3% of COPS hospitalized patient’s isolates, 26.7% of COPS RTE meat isolates and 25% of COPS food handler’s isolates. VanA, vanB, or both genes were detected in 64.7, 5.9, and 29.4% of all VAN-resistant isolates, respectively. The majority of the COPS isolates (50/60, 83.3%) have biofilm formation ability and harbored icaA (76%), icaD (74%), icaC (50%), and icaB (46%) biofilm-forming genes. The bap gene was not detected in any of the isolates. The ability of MRSA and VRSA isolates to produce biofilms in addition to being resistant to antimicrobials highlight the danger posed by these potentially virulent microorganisms persisting in RTE meat, food handlers, and patients. Taken together, good hygiene practices and antimicrobial surveillance plans should be strictly implemented along the food chain to reduce the risk of colonization and dissemination of MRSA and VRSA biofilm-producing strains.

The Fe3O4 nanoparticles functionalized by glutamic acid and conjugated with thiosemicarbazide decreases the expression of icaA and icaD biofilm genes in methicillin-resistant Staphylococcus aureus isolates

Biofilm production is an important survival strategy to increase resistance against antimicrobial agents. The icaA and icaD genes have been ascribed to play active roles for biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA). Therefore, preventing the expression of these genes is one of the most powerful strategies to control biofilm production. Today, nanoparticles offer a novel potential alternative for fighting resistant bacteria. Here, we have synthesized Fe3O4 magnetic nanoparticles (MNPs) that are functionalized by glutamic acid and conjugated with thiosemicarbazide using a chemical co-precipitation method and studied their effect on the biofilm-forming ability of MRSA.Diffuse Reflectance Spectroscopy (DRS), Fourier transform infrared spectroscopy (FTIR), scanning and transmission electron microscopy, Zeta potential analysis, Dynamic light scattering (DLS), energy dispersive X-Ray spectroscopy (EDS), and X-ray diffraction (XRD) used for validation and chemical characterization of synthesized nanoparticles.The obtained results confirmed the solution stability, good dispersion, and spherical/cubic morphology of Fe3O4@Glu–TSC MNPs with an average size of 35–60 nm. Minimum inhibitory concentration values of functionalized MNPs were reduced by two folds against each respected strain in comparison with the Fe3O4 MNPs (alone). Furthermore, the quantitative PCR data analysis showed a decrease in the expression of icaA and icaD genes in the presence of Fe3O4@Glu–TSC MNPs, up to 50% that was associated with a reduction of biofilm formation compared to control.The Fe3O4@Glu–TSC MNPs can be considered as a new inhibitor of biofilm production in antibiotic-resistant bacteria.

Description of Methicillin-Susceptible Staphylococcus aureus Clonal Complex 30 Related to the Pandemic Phage Type 80/81 Isolated from Patients in Three Tertiary Hospitals in Jos, North Central Nigeria

Objective: The prevalence of phage 80/81 Staphylococcus aureus strains, the pandemic strains that were dominant in the 1950s, had declined in the 1960s and 1970s. However, these strains have reemerged in some countries in recent years. This study investigated the antibacterial resistance, virulence, and the genetic backgrounds of CC30-MSSA isolates obtained from patients in three tertiary hospitals. Materials and Methods: Twenty-two CC30-MSSA isolates cultured from different clinical samples were investigated using antibiotic sensitivity testing, spa typing, multilocus sequence typing, and DNA microarray analysis. Results: All 22 isolates were susceptible to vancomycin (MIC ≤2 μg/mL), teicoplanin (MIC ≤2 μg/mL), and cefoxitin but were resistant to penicillin G (n = 22; 100.0%), tetracycline (n = 12; 54.5%), ciprofloxacin (n = 15; 68.2%), cadmium acetate (n = 22; 100%), mercuric chloride (n = 13; 59.1%), and ethidium bromide (n = 3; 13.6%). The isolates belonged to sequence type, ST30, and five spa types: t012 (n = 12; 54.5%), t019 (n = 5; 22.7%), t017 (n = 2; 9.1%), t037 (n = 2; 9.1%), and t318 (n = 1; 4.5%). All 22 isolates were positive for agrIII, cap8, clfA, clfB, icaA, icaC, icaD, cna, and staphylococcal enterotoxin gene clusters (seg, sei, sem, sen, seo, seu). Eight isolates carried lukS-PV and lukF-PV that code for Panton-Valentine leukocidin. Conclusion: The current CC30-MSSA isolates share phenotypic and genotypic characteristics with the pandemic phage 80/81 isolates that were common in the 1950s and 1960s. Continued surveillance is recommended to keep abreast of the changing epidemiology of S. aureus causing healthcare and community-associated infections.

Targeting Staphylococcus aureus and its biofilms with novel antibacterial compounds produced by Lactiplantibacillus plantarum SJ33.

The emergence of microbial resistance to conventional antibiotics, partially attributed to biofilm formation, has urged for new antimicrobial compounds. Here, we reported two novel low molecular weight (LMW) compounds from Lactiplantibacillus plantarum SJ33 and evaluated their biofilm inhibitory effects on Staphylococcus aureus. The compounds C1 and C2 were purified by RP-HPLC and structurally identified as 3-amino-5-hydroxy-6-(hydroxymethyl)-4-(1-hydroxyprop-2-yn-1-yl)-3,3a,4,5,6,7a-hexahydro-7H-indazol-7-one and 1-(dimethylamino)-3-hydroxy-3-((2-hydroxypropan-2-yl)oxy)-1-(methylamino)-butan-2-one by spectroscopic techniques. High ESI-MS data confirmed the molecular weight of C1 and C2 as 254.1141 and 234.1658Da, respectively. Time-kill assay demonstrated bactericidal action of compounds, whereas scanning electron microscopy revealed morphological changes in treated S. aureus MTCC96 and methicillin-resistant S. aureus (MRSA) cells. The antibacterial compounds reduced biofilm formation in S. aureus MTCC96 and MRSA by crystal violet assay. Further, fluorescence and scanning electron microscopic images exhibited biofilm formation by pathogens and biofilm inhibition by compounds treatment. The Quantitative RT PCR revealed the down-regulation of icaC and icaD genes involved in intercellular adhesion of biofilms. The results confirmed the anti-biofilm activity of novel LMW compounds by eliminating preformed biofilms formed by S. aureus MTCC96 and MRSA.

Comparison of the Frequency of Biofilm-Forming Genes (icaABCD) in Methicillin-Resistant S. aureus Strains Isolated from Human and Livestock.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause infections in both human and animal groups, which is a serious threat to public health worldwide. Attachment and colonization are the first steps for S. aureus pathogenesis, and biofilm-mediated infections have a significant negative impact on human and animal health. The MRSA can adapt to different environments and give rise to different strains of human and animal MRSA, causing transmissions of the disease between humans and animals. This study aimed to investigate biofilm production in vitro, and the presence of icaABCD genes in MRSA isolates in both human as well as the disease transmission between human and animal strains. In total, 39 human and 35 livestock isolates were evaluated by the Congo Red Agar method. The presence of mecA and icaABCDR genes were assessed by polymerase chain reaction (PCR), and finally, the PCR products were examined by agarose gel electrophoresis. The results showed that the mecA gene frequency in human and animal isolates was 64.1% and 36.1%, respectively, and there was a significant relationship between mecA and icaAD in human isolates. In addition, significant relationships were found between icaA and Rifampicin and also between icaC and Chloramphenicol and Penicillin in human isolates. In animal isolates, there was a significant relationship between mecA and Trimethoprim as well as between icaR and Rifampicin. It was concluded that all operon ica genes were involved in biofilm production, but icaA and icaD genes in MRSA were more closely associated with mecA. Both animal and human strains can be involved in disease transmission, but this conclusion should be made cautiously.

Clinical and Microbiological Profile of Diabetic Foot Ulcers Infected With Staphylococcus aureus in a Regional General Hospital in Bahia, Brazil.

It is necessary to know the resistance profile of Staphylococcus aureus to better control diabetic foot ulcer infections, to establish rational antibiotic therapy, and to avoid the development of resistant strains. This cross-sectional study evaluated the clinical parameters, virulence, and antimicrobial resistance profiles of S aureus in patients with diabetic foot disease admitted to a public hospital. S aureus strains were identified in patients with diabetes with amputation indication. Infected tissue samples were collected, microbes were isolated and identified. The microbial resistance profile was determined. Samples were also analyzed for biofilm formation and other virulence markers. The 34 individuals examined were mostly men, black, aged 60 years on average, and generally had a low income and education level. Most individuals had type 2 diabetes, and the mean time since diagnosis was 13.9 years. On an SF-36 (the Medical Outcomes Study 36-item short-form health survey) quality-of-life questionnaire, 75% of individuals obtained a score equal to 0 for physical impairment. S aureus specimens from 17 patients were isolated, corresponding to 50% of samples. Five isolates were classified as methicillin-resistant S aureus (MRSA). Molecular typing revealed that 20% of MRSA strains were SCCmec type V and 80% were type I. All isolates were sensitive to doxycycline; 61.5% were resistant to erythromycin, 38.5% to cefoxitin, 30.7% to clindamycin and ciprofloxacin, 23% to meropenem, 15.3% to gentamicin, 38.5% to oxacillin, and 7.7% (one strain) to vancomycin. Regarding biofilm production, 53% of samples were able to produce biofilms, and 84.6% had icaA and/or icaD genes. Additionally, the following enterotoxin genes were identified in the isolates: seb, sec, seg, and sei (5.9%, 5.9%, 11.8%, and 23.9%, respectively) and agr types 1 (5.9%) and 2 (11.8%). Genotypic evaluation made it possible to understand the pathogenicity of S aureus strains isolated from the diabetic foot; laboratory tests can assist in the monitoring of patients with systemic involvement.

Moringa oleifera seed oil extracted by pressurized n-propane and its effect against Staphylococcus aureus biofilms

ABSTRACT Staphylococcus aureus is often associated worldwide with foodborne illnesses, and the elimination of biofilms formed by this bacterium from industrial surfaces is very challenging. To date, there have been few attempts to investigate plant oils obtained by recent green technologies, applied against biofilms on usual surfaces of the food industry and bacteria isolated from such environment. Therefore, this study evaluated the activity of Moringa oleifera seed oil (MOSO), extracted with pressurized n-propane, against standard and environmental S. aureus biofilms. Additionally, a genotypic and phenotypic study of the environmental S. aureus was proposed. It was found that this bacterium was a MSSA (methicillin-sensitive S. aureus), a carrier of icaA and icaD genes that has strong adhesion (OD550=1.86 ± 0.19) during biofilm formation. The use of pressurized n-propane as a solvent was efficient in obtaining MOSO, achieving a yield of 60.9%. Gas chromatography analyses revealed the presence of a rich source of fatty acids in MOSO, mainly oleic acid (62.47%), behenic acid (10.5%) and palmitic acid (7.32%). On polystyrene surface, MOSO at 0.5% and 1% showed inhibitory and bactericidal activity, respectively, against S. aureus biofilms. MOSO at 1% allowed a maximum reduction of 2.38 log UFC/cm² of S. aureus biofilms formed on PVC (polyvinyl chloride) surface. Scanning electron microscopy showed disturbances on the surface of S. aureus after exposure to MOSO. These unprecedented findings suggest that MOSO extracted with pressurized n-propane is potentially capable of inhibiting biofilms of different S. aureus strains, thus, contributing to microbiological safety during food processing.

Effect of Ultraviolet Light on the Expression of icaD Gene in Staphylococcus aureus Local Isolates in Iraq.

The current study was designed to reveal the impact of ultraviolet (UV) light on polysaccharide intercellular adhesion of the icaD gene expression and its relation to biofilm as virulence markers in Staphylococcus aureus. In total, 37 isolates of S. aureus were gathered from various bacterial infection sources, and 26 S. aureus isolates were definite to icaD gene using the Polymerase Chain Reaction (PCR) technique. Prior to and after exposure to UV light, RNAs from the isolates were extracted which had strong biofilm formation by crystal violet staining assay, and they were then exposed to quantitative real-time PCR (qPCR). In vitro, the survival of isolates was evaluated after the exposition to several periods of the UV light power via counting the number of colonies. The results showed that the exposure to the UV light at 320 nm for 2, 4, 6, 8, and 10 min had inhibitory effects on the bacterial growth. The sub-lethal exposure time was 8 min. The rapid reduction of bacterial survival rates was associated with the increment irradiation time of UV power from 5 to 1.24 log10 CFU/ml. In general, down regulation of the icaD gene expression was decreased upon exposure to the UV light which was used as a physical agent for controlling the bacterial infection and biofilm formation.

Microbiological study of certain genes associated with biofilm forming capacity of Methicillin resistant Staphylococcus aureus in Egypt: An eye on Nifedipine repurposing

Background: Staphylococcus aureus remains one of the most prevalent pathogens associated with several infections. We aim to evaluate the biofilm forming capacity along with the presence of biofilm-associated genes in MRSA from surgical wound infections. In addition, potential antimicrobial activity of nifedipine was investigated. Methods: A total of 50 MRSA isolates were collected form surgical wound samples from clinical laboratories. The antimicrobial susceptibility and biofilm forming capacity were screened. PCR was used to detect icaA, icaD, hla, sirB, ebpS, fnbA, clfA, sdr and can genes. The antimicrobial and antibiofilm effect of nifedipine, alone and combined with levofloxacin, was determined. Preliminary molecular docking was employed to predict the binding affinity between nifedipine and different target proteins. Spa typing was performed to analyze MRSA strains. Results: All MRSA strains were multidrug-resistant and biofilm producers. The most abundant gene was hla (96%), followed by icaA and sirB with equal prevalence (88%). Biofilm formation was significantly associated with icaA, icaD, sdrE and sirB genes. In addition to the antibiofilm activity of nifedipine, there was a synergistic effect between it and levofloxacin, this finding was further given strength to by molecular docking where nifedipine had a binding affinity to HTH-type transcriptional regulator qacR. For the first time in Egypt, spa type t314 was reported. Conclusion: Nifedipine, alone and combined with levoflocaxin, showed promising results as antimicrobial and antibiofilm agent. Such effect might be due to efflux inhibition activity and worth additional investigation to understand the underlying mechanism.

Antiinfective properties of ursolic acid-loaded chitosan nanoparticles against Staphylococcus aureus.

The present study aimed to synthesize ursolic acid-loaded chitosan nanoparticles (UA-Ch-NPs) as an antiinfective agent against 21 Staphylococcus aureus isolates. The UA-Ch-NPs were synthesized by a simple method and then characterized by TEM, FTIR, DLS-zeta potential, and XRD analyses. According to the characterization results, highly dispersed spherical nanoparticles with a mean diameter of 258 nm and a zeta potential of + 40.1 mV were developed. The antibacterial properties of UA-Ch-NPs were investigated and their inhibitory effect on biofilm formation was demonstrated by AFM. Finally, the expression levels of icaA and icaD were measured using real-time PCR. Results indicated that the minimum inhibitory concentration (MIC) of UA and UA-Ch-NPs against S. aureus was 64 and 32 µg/mL, respectively. The treatment of bacterial cells with UA-Ch-NPs significantly decreased the expression of icaA and icaD genes which are engaged in biofilm formation. Our results indicated that UA-Ch-NPs could be a promising material for antibacterial and antibiofilm applications.