Ica Locus Research Articles

Various mutations in icaR, the repressor of the icaADBC locus, occur in mucoid Staphylococcus aureus isolates recovered from the airways of people with cystic fibrosis

Staphylococcus aureus is one of the major pathogens isolated from the airways of people with cystic fibrosis (pwCF). Recently, we described a mucoid S. aureus phenotype from respiratory specimens of pwCF, which constitutively overproduced biofilm that consisted of polysaccharide intercellular adhesin (PIA) due to a 5bp-deletion (5bp-del) in the intergenic region of the intercellular adhesin (ica) locus.Since we were not able to identify the 5bp-del in mucoid isolates of two pwCF with long-term S. aureus persistence and in a number of mucoid isolates of pwCF from a prospective multicenter study, these strains were (i) characterized phenotypically, (ii) investigated for biofilm formation, and (iii) molecular typed by spa-sequence typing. To screen for mutations responsible for mucoidy, the ica operon of all mucoid isolates was analyzed by Sanger sequencing. Whole genome sequencing was performed for selected isolates.For all mucoid isolates without the 5 bp-del, various mutations in icaR, which is the transcriptional repressor of the icaADBC operon. Mucoid and non-mucoid strains belonged to the same spa-type. Transformation of PIA-overproducing S. aureus with a vector expressing the intact icaR gene restored the non-mucoid phenotype.Altogether, we demonstrated a new mechanism for the emergence of mucoid S. aureus isolates of pwCF.

Quercetin's antibiofilm effectiveness against drug resistant Staphylococcus aureus and its validation by in silico modeling

Staphylococcus aureus is typically treated with antibiotics, however, due to its widespread and unselective usage, resistant strains of S. aureus have increased to a great extent. Treatment failure and recurring staphylococcal infections are also brought on by biofilm development, which boosts an organism's ability to withstand antibiotics and is thought to be a virulence factor in patients. The present study investigates the antibiofilm activity of naturally available polyphenol Quercetin against drug-resistant S. aureus. Micro dilution plating and tube adhesion methods were performed to evaluate the antibiofilm activity of quercetin against S. aureus. Quercetin treatment resulted in remarkably reduction of biofilm in S. aureus cells. Further we performed a study to investigate binding efficacies of quercetin with genes icaB and icaC from ica locus involved in biofilm formation. 3D structure of icaB, icaC and quercetin were retrieved from Protein data bank and PubChem chemical compound database, respectively. All computational simulation were carried out using AutoDock Vina and AutoDockTools (ADT) v 1.5.4. In silico study demonstrated a strong complex formation, large binding constants (Kb) and low free binding energy (ΔG) between quercetin and icaB (Kb = 1.63 × 10−5, ΔG = −7.2 k cal/mol) and icaC (Kb = 1.98 × 10−6, ΔG = −8.7 kcal/mol). This in silico analysis indicates that quercetin is capable of targeting icaB and icaC proteins which are essential for biofilm formation in S. aureus. Our study highlighted the antibiofilm activity of quercetin against drug resistant pathogen S.aureus.

In Vitro Inhibition of Growth, Biofilm Formation, and Persisters of Staphylococcus aureus by Pinaverium Bromide.

Biofilm or persister cells formed by Staphylococcus aureus are closely related to pathogenicity. However, no antimicrobials exist to inhibit biofilm formation or persister cells induced by S. aureus in clinical practice. This study found that pinaverium bromide had antibacterial activity against S. aureus, with the MIC50/MIC90 at 12.5/25 μM, respectively. Pinaverium bromide (at 4 × MIC) showed a rapid bactericidal effect on S. aureus planktonic cells, and it was more effective (at least 1-log10 cfu/mL) than linezolid, vancomycin, and ampicillin at 4 h of the time-killing test. Pinaverium bromide (at 10 × MIC) significantly inhibited the formation of S. aureus persister cells (at least 3-log10 cfu/mL) than linezolid, vancomycin, and ampicillin at 24, 48, 72, 96, and 120 h of the time-killing test. Biofilm formation and adherent cells of S. aureus isolates were significantly inhibited by pinaverium bromide (at 1/2 or 1/4 × MICs). The fluorescence intensity of the membrane polarity of S. aureus increased with the treatment of pinaverium bromide (≥1 × MIC), and the MICs of pinaverium bromide increased by 4 times with the addition of cell membrane phospholipids, phosphatidyl glycerol and cardiolipin. The cell viabilities of human hepatocellular carcinoma cells HepG2 and Huh7, mouse monocyte-macrophage cells J774, and human hepatic stellate cells LX-2 were slightly inhibited by pinaverium bromide (<50 μM). There were 54 different abundance proteins detected in the pinaverium bromide-treated S. aureus isolate by proteomics analysis, of which 33 proteins increased, whereas 21 proteins decreased. The abundance of superoxide dismutase sodM and ica locus proteins icaA and icaB decreased. While the abundance of global transcriptional regulator spxA and Gamma-hemolysin component B increased. In conclusion, pinaverium bromide had an antibacterial effect on S. aureus and significantly inhibited the formation of biofilm and persister cells of S. aureus.

Review on Molecular Cross-talk of Biofilm Producing Mechanisms of Staphylococcus aureus

Here the review converses the `molecular cross-talk` of biofuel production mechanisms for Staphylococcus aureus. Staphylococcus aureus is a leading cause of bacterial infections globally in both healthcare and community settings. The succes of this bacterium is the of an expansive repertoire of virulence factors in combination with acquired antibiotic resistance and propensity for biofilm formation. S. aureus leverages these factors to adapt to and subvert the host immune response. With the burgeoning fiels of immunometabolism, is has become clear that the metabolic program of leukocytes dictates their inflammatory status and overall effectiveness is clearing an infection. The treatment of S. aureus infections become complicated due to the capacity of S. aureus �multidrug-resistant� occurs because of biofilm formationon the surfaces depending on biotic and abiotic factors, genetic factors, and numerous environmental, which vary from species to species. A broad range of molecular phenomenon contributes a high range of recalcitrance that is insisting on the biofilm formation. The previous published literature illustrated that all strains of Staphylococcal sp. contain the �ica locus� and several can form biofilms in vitro condition. Absences of �ica locus� results diminish of capability to produce biofuels, along with `PIA gene`, or mediate `N-acetyl glucosaminyl transferase activity� in vitro condition.

The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation.

Staphylococcus epidermidis is a common cause of device related infections on which pathogens form biofilms (i.e., multilayered cell populations embedded in an extracellular matrix). Here, we report that the transcription factor SpoVG is essential for the capacity of S. epidermidis to form such biofilms on artificial surfaces under in vitro conditions. Inactivation of spoVG in the polysaccharide intercellular adhesin (PIA) producing S. epidermidis strain 1457 yielded a mutant that, unlike its parental strain, failed to produce a clear biofilm in a microtiter plate-based static biofilm assay. A decreased biofilm formation capacity was also observed when 1457 ΔspoVG cells were co-cultured with polyurethane-based peripheral venous catheter fragments under dynamic conditions, while the cis-complemented 1457 ΔspoVG::spoVG derivative formed biofilms comparable to the levels seen with the wild-type. Transcriptional studies demonstrated that the deletion of spoVG significantly altered the expression of the intercellular adhesion (ica) locus by upregulating the transcription of the ica operon repressor icaR and down-regulating the transcription of icaADBC. Electrophoretic mobility shift assays (EMSA) revealed an interaction between SpoVG and the icaA-icaR intergenic region, suggesting SpoVG to promote biofilm formation of S. epidermidis by modulating ica expression. However, when mice were challenged with the 1457 ΔspoVG mutant in a foreign body infection model, only marginal differences in biomasses produced on the infected catheter fragments between the mutant and the parental strain were observed. These findings suggest that SpoVG is critical for the PIA-dependent biofilm formation of S. epidermis under in vitro conditions, but is largely dispensable for biofilm formation of this skin commensal under in vivo conditions.

Cuminum cyminum L. Essential Oil: A Promising Antibacterial and Antivirulence Agent Against Multidrug-Resistant Staphylococcus aureus.

Cuminum cyminum L. (cumin) is valued for its aromatic and medicinal properties. There are several reports of antibacterial activity of C. cyminum essential oil (CcEO). Accordingly, the present study was conducted to investigate the mechanism(s) of action of the CcEO against multidrug-resistant (MDR) Staphylococcus aureus. Therefore, 10 S. aureus MDR isolates, obtained from different sources, were selected based on the antibiotic susceptibility patterns and the Clinical and Laboratory Standards Institute definition and subjected to the examinations. Our results exhibited promising bacteriostatic and bactericidal properties of the CcEO. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration values ranged from 5 to 10 and 10 to 20 μL ⋅ mL–1, respectively. Scanning electron microscope was used to assess the bacterial cell structure and morphology after the induction with 1/2 MIC concentration of the CcEO. The observed morphological changes appeared to be deformation of the cell membrane and destruction of the cells. In the case of quorum sensing inhibitory potential, treatment of S. aureus isolates with the sub-MIC concentrations (1/2 MIC) of the CcEO significantly reduced the hld expression (3.13-fold downregulation), which considerably controls S. aureus quorum-sensing accessory regulator system. Another virulence factor influenced by the CcEO was the polysaccharide intercellular adhesion production system, as an important component of cell–cell adhesion and biofilm formation. Consequently, the expression level of the intercellular adhesion (ica) locus in the S. aureus cells was examined following treatment with CcEO. The results showed significant decrease (−3.3-fold) in ica expression, indicating that the CcEO could potentially interfere with the process of biofilm formation. Using the ethidium bromide efflux inhibition assay, the S. aureus NorA efflux pump was phenotypically but not genotypically (in quantitative polymerase chain reaction assay) affected by the CcEO treatment. Using gas chromatography–mass spectrometry analysis, cuminic aldehyde (38.26%), α,β-dihydroxyethylbenzene (29.16%), 2-caren-10-al (11.20%), and γ-terpinene (6.49%) were the most detected compounds. The antibacterial and antivirulence action of the CcEO at sub-MIC concentrations means that no microbial resistance will be promoted and developed after the treatment with this agent. These findings revealed that the CcEO is a promising antibacterial agent to control infections caused by the MDR S. aureus strains.

Evaluation of the role of staphylococci in the pathomechanism of conjunctivitis

PurposeDetermination of the association between ica genes and phenotypic biofilm formation in staphylococcal isolates involved in conjunctivitis, their antibiotic resistance as well as detection of selected virulence characteristics: adhesion to epithelial cells and in vitro cytotoxicity.MethodsThe study included 26 Staphylococcus aureus (SA) and 26 Staphylococcus epidermidis (SE) isolates. The presence of icaAD genes and ica operon was determined by the PCR assay. Phenotypic biofilm formation was verified using the microtitre plate assay. Antibiotic resistance was performed using the disc diffusion method. Staphylococcal ability to attach to host cells was assessed by flow cytometry. Cytotoxicity on epithelial cells was evaluated by LDH assay.ResultsThe ica genes were detected in 26.9% of SE and in 42.3% of SA isolates. Only 15.3% of isolates (SE) were positive for both the icaAD and the ica operon. Phenotypically, 19.2% of SE isolates were strong biofilm producers, among which three were both icaAD- and ica operon-positive. About 26.9% of SA isolates were strong biofilm producers. Methicillin resistance (MR) was detected in 34.6% of SE and 26.9% of SA isolates. About 75% of MR isolates were multidrug resistant. SA isolates adhered to host cells more extensively than SE. SA isolates released higher level of LDH than SE.ConclusionsAdherence abilities were commonly observed in staphylococci associated with conjunctivitis. However, low prevalence of isolates positive for a complete and functional ica locus and low prevalence of strong biofilm producers was detected. SA adhered to a greater extent to eukaryotic cells than SE and were more cytotoxic.

Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens.

Quercetin Inhibits Biofilm Formation by Decreasing the Production of EPS and Altering the Composition of EPS in Staphylococcus epidermidis

Staphylococcus epidermidis is an opportunistic pathogen, and its biofilm formation ability is an important virulent factor. Quercetin, a typical flavonoid ubiquitously used in dietary supplementation, is known for its antioxidant property, but its anti-biofilm activity against S. epidermidis remains unknown. In this study, the anti-biofilm activity of quercetin was investigated using S. epidermidis ATCC35984, a strong biofilm-positive strain. An attempt was made to disclose the mechanisms of the anti-biofilm activity of quercetin. S. epidermidis exhibited a less cell surface hydrophobicity after quercetin treatment. Also, quercetin effectively inhibited S. epidermidis cells from adhering to the glass slides. Quercetin downregulated the intercellular adhesion (ica) locus and then polysaccharide intercellular adhesin (PIA) production was reduced. Therefore, S. epidermidis cells became less hydrophobic, which supported quercetin’s anti-biofilm effect. Our study suggests that quercetin from plants be given further attention as a potential anti-biofilm agent against the biofilm formation of S. epidermidis, even biofilm infections of other bacteria.

Biofilm production, antibiotic resistance and the presence of ica, bap, agr and blaz genes in bovine mastitis-associated Staphylococcus aureus isolates from Karnataka

Biofilm formation is an important virulence determinant of Staphylococcus aureus which is a major etiological agent of bovine mastitis. Here, 132 bovine mastitis-associated S. aureus were subjected to biofilm production, antimicrobial susceptibility, and the detection of ica, bap, agr and blaZ genes. It was found that 33.3% of the isolates produced biofilm. The number of isolates resistant to individual antibiotics increased by 1.2- to 7.0-fold when growing in biofilm versus planktonic mode of growth, and the spectrum of antibiotics as well as the number of isolates resistant to various antibiotics increased with the increase in the density of the biofilm. However, there was no correlation between the strength of biofilm and the extent of antibiotic resistance. When evaluated for the presence of genes reported to be associated with biofilm formation, bap gene was detected in a significant number (12.9%) of the isolates, while the ica locus was equally distributed among strains that produced biofilm or not. A significant correlation was observed between the presence of agrI and ica genes. High blaZ positivity was observed irrespective of penicillin resistance or biofilm phenotype. In summary, biofilm forming S. aureus was more resistant to many antibiotics in biofilm than in planktonic form, and none of the genes studied showed association with phenotypic studies of either biofilm production or antimicrobial susceptibility. The study highlights the need for further investigations on the inter-relationships between biofilm, antibiotic resistance and associated genotypes.

Streptomyces-derived actinomycin D inhibits biofilm formation via downregulating ica locus and decreasing production of PIA in Staphylococcus epidermidis.

The objective of this study was to investigate the biofilm inhibitory activity of Streptomyces-derived actinomycin D against biofilm formation by Staphylococcus epidermidis. The microtitre plate method and microscopy were used to detect the biofilm formation of S. epidermidis. And an attempt was made to detect the effect of actinomycin D on important biofilm components, exopolysaccharides (EPS) in S. epidermidis using precolumn derivation HPLC. Also cell surface hydrophobicities of S. epidermidis were assessed to explore action mechanisms. The qPCR was performed to demonstrate the genetic mechanisms of biofilm formation by S. epidermidis. Unlike other antibiotics, actinomycin D (1·5μgml-1 ) from Streptomyces luteus significantly inhibited biofilm formation by S. epidermidis. Additionally, it effectively inhibited S. epidermidis cells from adhering to glass slides. Actinomycin D downregulated ica locus and then the reduced polysaccharide intercellular adhesin production caused S. epidermidis cells to become less hydrophobic, thus supporting its anti-biofilm effect. Streptomyces-derived actinomycin D is active in inhibiting the biofilm formation of S. epidermidis. Actinomycin D can be used as a promising antibiofilm agent in inhibiting S. epidermidis biofilm formation. The study is also the first insight into how actinomycin D inhibited the biofilm formation of S. epidermidis. Actinomycin D could potentially be used to reduce the risk of biofilm-associated infections. Our study also suggests that the metabolites from Actinomycete strains keep further attention as potential antibiofilm agents against biofilm formation of S. epidermidis, even biofilm infections of the other bacteria.

Relationship between biofilm gene expression with antimicrobial resistance pattern and clinical specimen type based on sequence types (STs) of methicillin-resistant S. aureus.

The ica genes in methicillin-resistant Staphylococcus aureus (MRSA) play an important role in biofilm formation. The aim of this study is to define effect of antibiotic resistance and clinical specimens to the expression of ica genes based on their sequence types (STs) and clonal complex (CC). One-hundred (100) S. aureus strain were collected from two teaching therapeutic centers in Hamedan, Iran. Then, the PCR, qPCR, and MLST were used to characterize strains. The results indicated that 29 (29%), 15 (15%), and 5 (5%) strain were strong, mediate, weak biofilm producer, respectively, and the icaA (17%) and icaC (14%) genes were the most abundant. However, two unique STs (3667, 491) in Iran were reported and ST30 and ST11 were the most abundant STs and CC30 and CC5 were observed among MRSA and MSSA strains. High activity in ica locus was observed among strains collected from wound and catheter strains. Also, expression level of icaA gene increased in all strains except ST30 and ST491. Moreover, the highest expression level was observed in CC1, CC7, and CC11. Likewise, activity of the icaC gene was only observed in CC5. Furthermore, the expression of all ica genes in CC5 was significantly correlated with the type of biofilm and the clinical sample. In this study demonstrated that the frequency distribution of STs and CCs in different strains of MRSA was higher than methicillin-sensitive strains. Also, the type of clinical specimen and expression of ica genes played an important role in this abundance.

A non-coding RNA from the intercellular adhesion (ica) locus of Staphylococcus epidermidis controls polysaccharide intercellular adhesion (PIA)-mediated biofilm formation.

Polysaccharide intercellular adhesin (PIA)-associated biofilm formation is mediated by the intercellular adhesin (ica) locus and represents a major pathomechanism of Staphylococcus epidermidis. Here, we report on a novel long non-coding (nc)RNA, named IcaZ, which is approximately 400 nucleotides in size. icaZ is located downstream of the ica repressor gene icaR and partially overlaps with the icaR 3' UTR. icaZ exclusively exists in ica-positive S. epidermidis, but not in S. aureus or other staphylococci. Inactivation of the gene completely abolishes PIA production. IcaZ is transcribed as a primary transcript from its own promoter during early- and mid-exponential growth and its transcription is induced by low temperature, ethanol and salt stress. IcaZ targets the icaR 5' UTR and hampers icaR mRNA translation, which alleviates repression of icaADBC operon transcription and results in PIA production. Interestingly, other than in S. aureus, posttranscriptional control of icaR mRNA in S. epidermidis does not involve icaR mRNA 5'/3' UTR base pairing. This suggests major structural and functional differences in icaADBC operon regulation between the two species that also involve the recruitment of ncRNAs. Together, the IcaZ ncRNA represents an unprecedented novel species-specific player involved in the control of PIA production in NBSP S. epidermidis.

Clonally Diverse Methicillin and Multidrug Resistant Coagulase Negative Staphylococci Are Ubiquitous and Pose Transfer Ability Between Pets and Their Owners.

Sixty-eight owners and 66 pets, from 43 unrelated pet-owning households were screened for methicillin-resistant coagulase negative staphylococci (MRCoNS), potential cases of MRCoNS interspecies transmission (IT), and persistence. MRCoNS isolates were identified by microbiological and molecular tests. MLST-based phylogenetic analysis was performed in Staphylococcus epidermidis isolates. Antimicrobial susceptibility was evaluated using phenotypic and molecular methods. SCCmec type and the presence of biofilm-related ica locus was PCR-tested. Isolates suspected for MRCoNS IT cases were subjected to SmaI-PFGE analysis and individuals from positive households were followed-up for 1 year for carriage dynamics (every 3 months, T0–T4). Nineteen MRCoNS isolates from owners (27.9%) and 12 from pets (16.7%) were detected, coming from 20 households (46.5%). S. epidermidis was predominant (90 and 67% of human and animal strains, respectively), showing high phylogenetic diversity (16 STs among 24 strains). Methicillin-resistant S. epidermidis (MRSE) strains belonged to CC5 (75%), CC11 (12.5%), singleton S556 (8.3%), and S560 (4.17%). Significant host-associated differences were observed for resistance to aminoglycosides, co-trimoxazole, chloramphenicol (higher in animal isolates) and tetracycline (higher among human strains). Multidrug resistance (MDR) was common (68.4%) and associated with human strains. Great diversity of ccr and mec complexes were detected, most strains being non-typeable, followed by SCCmecIV and V. Over one third of isolates (most from owners), carried the ica locus, all MRSE CC5. Two sporadic IT cases (T0) were identified in owners and dogs from two households (4.7%), with diverse interspecies-exchanged clones detected along the sampling year, especially in dogs. A comparative analysis of all MRCoNS, with all nasal coagulase positive staphylococci (CoPS) recovered from the same individuals at T0, revealed that CoPS alone was predominant in owners and pets, followed by co-carriage of CoPS and MRCoNS in owners but single MRCoNS in pets. Statistical analyses revealed that owners are more prone to co-carriage and that co-existence of IT cases and co-carriage are positively interrelated. MRCoNS from healthy owners and their pets are genetically heterogeneous MDR strains that are spread in the community. Therefore, pets also contribute to the dissemination of successful human clones. Owner-pet inhabitancy increases the risk for staphylococcal temporal concomitance with its subsequent risk for bacterial infection and genetic exchange.

2307. Use of Whole-Genome Sequencing to Determine Adhesin and Biofilm-Associated Gene Profiles Among Pediatric Staphylococcus aureus Device-Related Infection Isolates Compared With Skin and Soft-Tissue Infection Isolates

BackgroundAdhesins or microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) and the ica locus help mediate S. aureus adherence to host tissue and biofilm formation and are thought to play important roles in the pathogenesis of device related infections (DRIs). We hypothesized that S. aureus isolates from pediatric DRIs differ in MSCRAMM and biofilm-associated gene profiles and display greater strain diversity compared with skin and soft-tissue infection (SSTI) isolates.MethodsPatients and isolates were identified from a prospective S. aureus surveillance study at Texas Children’s Hospital, 2008–2016. Clinical data were collected retrospectively. Age and date of infection matched SSTI control isolates were selected 4:1. Isolates were genotyped by pulsed-field gel electrophoresis. Whole genome sequencing was performed (Illumina MiSeq). Data were analyzed with CLC Genomics Workbench for the presence of MSCRAMMS (clfA, clfB, ebh, fbp, fnbpA, fnbpB, isdA, isdB, sdrC, sdrD, sdrE), biofilm-associated genes (icaA,D,B,C), accessory gene regulator group, and by multilocus-sequence typing (MLST) with eBurst analysis (www.phyloviz.net). Conditional logistic regression and Fisher’s exact were used for analysis (STATA11).ResultsForty-five patients with 47 DRIs were identified (Table 1). Isolates from 47 DRIs and 188 SSTIs were analyzed for the presence of MSCRAMM and biofilm-associated genes. clfA, clfB, fbp, isdA, isdB, and icaA,D,B,C were present among DRIs and SSTIs more than 98% of the time. Isolates from DRIs or SSTIs did not differ significantly in carriage of MSCRAMMs or the ica locus. DRIs were MSSA (34, 72%), nonUSA300 (39, 83%), and belonged to 19 sequence types (STs). SSTIs were MSSA (79, 42%), nonUSA300 (57, 30%), and belonged to 39 STs (Table 2). Among DRI isolates, STs 5 and 8 were most common (23% each, Figure 1). SSTI isolates were predominately ST8 (68%).Conclusion S. aureus isolates from DRIs were significantly more likely to be MSSA and nonUSA300 (P < 0.0001 for both) compared with SSTIs. The majority of S. aureus isolates harbored all MSCRAMM and biofilm-associated genes analyzed. Evaluating genetic polymorphisms and gene expression profiles may clarify the role of adhesion genes in the pathogenesis of DRIs vs. SSTIs. Disclosures All authors: No reported disclosures.

Penicillin-resistant characterization of Staphylococcus aureus isolated from bovine mastitis in Gansu, China

AbstractBovine mastitis caused by Staphylococcus aureus is difficult to treat because of increasing resistance against antibiotics, especially penicillin. β-Lactamase and biofilm are responsible for penicillin resistance of S. aureus. The aim of this study was to investigate the β-lactamase activity and biofilm formation capacity of 37 penicillin-resistant S. aureus strains (35 were blaZ positive and 2 were blaZ negative) from bovine mastitis in Gansu Province, China, as well as to measure the intercellular adhesion genes icaA and icaD of these strains. β-Lactamase Test Kit was used to determine the β-lactamase activity, biofilm formation was tested by semi-quantitative adherence assay method. Moreover, the presence of icaA and icaD were measured by PCR. A total of 32 penicillin-resistant S. aureus strains, including the two blaZ-negative strains, were identified as β-lactamase producers. All tested S. aureus isolates produced biofilm in the microtiter plate assay. Meanwhile, all these strains were PCR-positive for the ica locus, icaA and icaD. The study indicated high prevalence of β-lactamase activity, biofilm-forming capacity, and the ica genes among the penicillin-resistant S. aureus isolates, and implied that S. aureus resistant to penicillin was attributed to multiple mechanisms.

Suppression of biofilm formation in Staphylococcus aureus by targeting Staphylococcal accessory regulator X expression using siHybrids

Nosocomial infections are becoming increasingly prevalent due to the ability of bacteria to adhere to implanted medical devices. Over 600,000 prosthetic joint replacements are performed yearly in the United States, with approximately 2% of those cases developing infections. The implantation of cardiac valves has increased by 42%, but with a corresponding 210% increased infection rate of Staphylococcus gram‐positive bacteria. S. aureus accounts for up to 73% of these infections. The ability of bacteria to adhere to the hard surfaces of medical devices is due to production of a biofilm, which is initiated by the secretion of polysaccharide intercellular adhesion (PIA) molecules. In Staphylococcal species, PIA production and regulation is controlled by the icaADBC (ica) operon, a sequence of four genes that are all involved in the production of PIA molecules. The icaA gene produces an N‐acetyl‐glucosaminyltransferase like protein, with the icaD gene product enhancing its activity. The icaC gene encodes a membrane protein that initiates the production of longer chains of polysaccharides and their translocation to the cell surface, while the icaB product is responsible for deacetylating the poly‐N‐acetylglucosamine ica transcript.The ica locus has several known activators and repressors. One of the best‐characterized factors is the staphylococcal accessory regulator A (sarA), which functions as an activator of the locus. Research has shown that when sarA transcripts are suppressed, biofilm growth is repressed. SarX is closely homologous to SarA and is believed to share similar, activating functions; however, the product of the sarX locus is not as well characterized. We investigated if sarX function can be suppressed through the use of sequence specific siHybrids, and if so, whether suppressing sarX has a repressing effect on biofilm development in Staphylococcus aureus. We hypothesized that suppression of sarX will cause repression of biofilm formation. S. aureus was passively transformed with siHybrids and the resulting biofilm growth was measured using crystal violet staining. RNA was isolated from transformed bacteria to determine if levels of sarX transcripts had changed. SiHybrid treatment groups were compared to three controls: an untreated S. aureus positive control, a sterile media negative control, and an E. coli negative control. The results indicated that repressing biofilm growth through the suppression of sarX transcripts with siHybrids led to a seventeen to forty percent decrease in biofilm formation. An ANOVA with a p‐value of 0.5 was used to determine significance. Inhibition of S. aureus at the molecular level, by targeting the RNA transcripts of the icaADBC activator, sarX, presents a novel treatment for nosocomial infections.

A Novel Repressor of the ica Locus Discovered in Clinically Isolated Super-Biofilm-Elaborating Staphylococcus aureus.

ABSTRACTStaphylococcus aureus TF2758 is a clinical isolate from an atheroma and a super-biofilm-elaborating/polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosamine (PNAG)-overproducing strain (L. Shrestha et al., Microbiol Immunol 60:148–159, 2016, https://doi.org/10.1111/1348-0421.12359). A microarray analysis and DNA genome sequencing were performed to identify the mechanism underlying biofilm overproduction by TF2758. We found high transcriptional expression levels of a 7-gene cluster (satf2580 to satf2586) and the ica operon in TF2758. Within the 7-gene cluster, a putative transcriptional regulator gene designated rob had a nonsense mutation that caused the truncation of the protein. The complementation of TF2758 with rob from FK300, an rsbU-repaired derivative of S. aureus strain NCTC8325-4, significantly decreased biofilm elaboration, suggesting a role for rob in this process. The deletion of rob in non-biofilm-producing FK300 significantly increased biofilm elaboration and PIA/PNAG production. In the search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by rob, we identified open reading frame (ORF) SAOUHSC_2898 (satf2584). Our results suggest that ORF SAOUHSC_2898 (satf2584) and icaADBC are required for enhanced biofilm elaboration and PIA/PNAG production in the rob deletion mutant. Rob bound to a palindromic sequence within its own promoter region. Furthermore, Rob recognized the TATTT motif within the icaR-icaA intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in S. aureus. Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is an important regulator of biofilm elaboration through its control of SAOUHSC_2898 (SATF2584) and Ica protein expression in S. aureus.

Detection of intercellular adhesion icaAD genes in Staphylococcus aureus and coagulase-negative staphylococci and their role in biofilm production

Objectives This study aimed to investigate the resistance patterns of nosocomial and community-acquired Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and screen methicillin-resistant and vancomycin-resistant isolates by phenotypic and genotypic methods. It also aimed to determine the biofilm-forming capacity and detect icaA and icaD genes responsible for polysaccharide intercellular adhesion synthesis and analyze the association between the biofilm-forming capacity of staphylococcal isolates and their multidrug resistance patterns. Background Biofilms have dramatically increased resistance to antibiotics. The genes and products of ica locus [icaR (regulatory) and icaADBC (biosynthetic) genes] have been demonstrated to be necessary for biofilm formation and virulence. Materials and methods The study was carried out on specimens collected from Menoufia University Hospitals. Isolation, identification, and antimicrobial susceptibility of staphylococcal isolates were carried out using standardized microbiological methods. Phenotypic biofilm detection was carried out by microtiter plate adherence assay, the Congo red agar method, and the modified Congo red agar method. All clinical isolates (CIs) of S. aureus and CoNS demonstrating reduced susceptibility to methicillin and vancomycin and showing the ability to form biofilm were tested for the presence of methicillin-resistant gene (mecA), vancomycin-resistant genotypes (vanA and vanB), and biofilm-producing genes (icaA and icaD) by means of multiplex PCR. Results About 82.4% of S. aureus isolates were methicillin-resistant S. aureus, whereas only 76.5% of them were positive for the mecA gene. VanA-positive gene was detected in 10.3% of S. aureus isolates. Regarding CoNS isolates, 76.9% were negative for the mecA gene and 78.8% were methicillin-resistant coagulase-negative staphylococci. About 10% were positive for the vanA genes in CoNS isolates. Biofilm formation was detected in 45.6 and 41.2% of S. aureus and in 76.9 and 55.8% of CoNS isolates, as detected by microtiter plate and Congo red agar, respectively. Ica genes were detected in 38.2% of S. aureus CIs and in 63.5% of CoNS CIs. Conclusion The biofilm-forming ability of staphylococcal isolates correlated with clinical significance and drug resistance. Biofilm-forming ability in the absence of icaA and icaD genes highlights the importance of further genetic investigations of ica- independent biofilm formation mechanisms.

Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects

Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection.