Nanoparticle ultrasonication outperforms conventional irrigation solutions in eradicating Staphylococcus aureus biofilm from titanium surfaces: an in vitro study.

PURPOSE: Bacterial biofilms create a challenge in the treatment of prosthetic joint infection (PJI), and failure to eradicate biofilms is often implicated in the high rates of recurrence. In this study, we aimed to compare the effectiveness of a novel nanoparticle ultrasonication technology on Staphylococcus aureus biofilm eradication compared to commonly used orthopedic irrigation solutions. METHODS: Twenty-four sterile, titanium alloy discs were inoculated with a standardized concentration of methicillin-resistant S. aureus and cultured for seven days to allow for biofilm formation. Discs were then treated with either ultrasonicated nanoparticle therapy or irrigation with chlorhexidine gluconate, povidone-iodine or normal saline. The remaining bacteria on each surface was subsequently plated for colony-forming units of S. aureus. Bacterial eradication was reported as a decrease in CFUs relative to the control group. Mann-Whitney U tests were used to compare between groups. RESULTS: Treatment with ultrasonicated nanoparticles resulted in a significant mean decrease in CFUs of 99.3% compared to controls (p < 0.0001). Irrigation with povidone-iodine also resulted in a significant 77.5% reduction in CFUs compared to controls (p < 0.0001). Comparisons between ultrasonicated nanoparticles and povidone-iodine demonstrated a significantly higher reduction in bacterial CFUs in the nanoparticle group (p < 0.0001). CONCLUSION: Ultrasonicated nanoparticle were superior to commonly used bactericidal irrigation solutions in the eradication of S. aureus from a titanium surface. Future clinical studies are warranted to evaluate this ultrsonication technology in the treatment of PJI.

Promiscuous signaling by a regulatory system unique to the pandemic PMEN1 pneumococcal lineage.

Streptococcus pneumoniae (pneumococcus) is a leading cause of death and disease in children and elderly. Genetic variability among isolates from this species is high. These differences, often the product of gene loss or gene acquisition via horizontal gene transfer, can endow strains with new molecular pathways, diverse phenotypes, and ecological advantages. PMEN1 is a widespread and multidrug-resistant pneumococcal lineage. Using comparative genomics we have determined that a regulator-peptide signal transduction system, TprA2/PhrA2, was acquired by a PMEN1 ancestor and is encoded by the vast majority of strains in this lineage. We show that TprA2 is a negative regulator of a PMEN1-specific gene encoding a lanthionine-containing peptide (lcpA). The activity of TprA2 is modulated by its cognate peptide, PhrA2. Expression of phrA2 is density-dependent and its C-terminus relieves TprA2-mediated inhibition leading to expression of lcpA. In the pneumococcal mouse model with intranasal inoculation, TprA2 had no effect on nasopharyngeal colonization but was associated with decreased lung disease via its control of lcpA levels. Furthermore, the TprA2/PhrA2 system has integrated into the pneumococcal regulatory circuitry, as PhrA2 activates TprA/PhrA, a second regulator-peptide signal transduction system widespread among pneumococci. Extracellular PhrA2 can release TprA-mediated inhibition, activating expression of TprA-repressed genes in both PMEN1 cells as well as another pneumococcal lineage. Acquisition of TprA2/PhrA2 has provided PMEN1 isolates with a mechanism to promote commensalism over dissemination and control inter-strain gene regulation.

Tonsillolith: not just a stone but a living biofilm.

OBJECTIVE: To study the morphology and activity of tonsilloliths, demonstrating oxygen respiration, denitrification, and acidification on exposure to sucrose. STUDY DESIGN: Tonsilloliths were extracted in atraumatic conditions during tonsillectomy from 16 adults and sent to two different laboratories for histological, bacteriological, and biofilm studies under sterile conditions. SETTING: Multicenter laboratory study. SUBJECTS AND METHODS: Multiple tonsilloliths from two patients examined by confocal microscopy and microelectrodes were used to measure aerobic/anaerobic respiration and acid production (dissolved oxygen, nitrous oxide, pH) when exposed to saliva following addition of sucrose and fluoride. RESULTS: Morphologically, tonsilloliths were similar to dental biofilms, containing corncob structures, filaments, and cocci. Microelectrodes showed that the microorganisms respired oxygen and nitrate. The oxygen concentration in the center of the tonsillolith was depleted to approximately one-tenth of that of the overlying fluid. The addition of sucrose resulted in acid production within the tonsillolith, dropping the pH from 7.3 to 5.8. The data showed stratification with oxygen respiration at the outer layer of tonsillolith, denitrification toward the middle, and acidification toward the bottom. The depletion of oxygen and acid production following addition of sucrose may allow the proliferation of anaerobic/acidophilic bacteria. Fluoride suppressed acid production in the presence of sucrose. CONCLUSIONS: Tonsilloliths exhibit biofilm structure and the formation of chemical gradients through physiological activity. Although tonsillectomy is an option for treating cryptic infections, understanding the morphology and biofilm characteristics of tonsilloliths may stimulate scientists to use limited or targeted remedies in the future.

Resistance of Synthetic and Biologic Surgical Meshes to Methicillin-Resistant Staphylococcus aureus Biofilm: An In Vitro Investigation.

Surgical meshes have become the standard procedure for a variety of surgical applications with 20 million meshes being implanted each year. The popularity of mesh usage among surgeons is backed by the multiple studies that support its functionality as a tool for improving surgical outcomes. However, their use has also been associated with infectious surgical complications and many surgeons have turned to biologic meshes. While there have been several studies investigating synthetic meshes, there is limited data comparing synthetic and biologic meshes in vitro in an infection model. This study evaluates the in vitro susceptibility of both synthetic and biologic meshes to single-species methicillin-resistant Staphylococcus aureus (MRSA) biofilms. This research compares biofilm biomass, average thickness, and coverage between the three meshes through florescent in situ hybridization (FISH), confocal scanning microscopy (CSLM), and image analysis. We also report the varying levels of planktonic and attached bacteria through sonication and cfu counts. While the data illustrates increased biofilm formation on biologic mesh in vitro, the study must further be investigated in vivo to confirm the study observations.

Deletion of genes involved in the ketogluconate metabolism, Entner-Doudoroff pathway, and glucose dehydrogenase increase local and invasive virulence phenotypes in Streptococcus pneumoniae.

Streptococcus pneumoniae displays increased resistance to antibiotic therapy following biofilm formation. A genome-wide search revealed that SP 0320 and SP 0675 (respectively annotated as 5-keto-D-gluconate-5-reductase and glucose dehydrogenase) contain the highest degree of homology to CsgA of Myxococcus xanthus, a signaling factor that promotes cell aggregation and biofilm formation. Single and double SP 0320 and SP 0675 knockout mutants were created in strain BS72; however, no differences were observed in the biofilm-forming phenotypes of mutants compared to the wild type strain. Using the chinchilla model of otitis media and invasive disease, all three mutants exhibited greatly increased virulence compared to the wild type strain (increased pus formation, tympanic membrane rupture, mortality rates). The SP 0320 gene is located in an operon with SP 0317, SP 0318 and SP 0319, which we bioinformatically annotated as being part of the Entner-Doudoroff pathway. Deletion of SP 0317 also resulted in increased mortality in chinchillas; however, mutations in SP 0318 and SP 0319 did not alter the virulence of bacteria compared to the wild type strain. Complementing the SP 0317, SP 0320 and SP 0675 mutant strains reversed the virulence phenotype. We prepared recombinant SP 0317, SP 0318, SP 0320 and SP 0675 proteins and confirmed their functions. These data reveal that disruption of genes involved in the degradation of ketogluconate, the Entner-Doudoroff pathway, and glucose dehydrogenase significantly increase the virulence of bacteria in vivo; two hypothetical models involving virulence triggered by reduced in carbon-flux through the glycolytic pathways are presented.

Identification and Characterization of msf, a Novel Virulence Factor in Haemophilus influenzae.

Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi) emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs). Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf) and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4) (KO) resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites.

Temporal expression of agrB, cidA, and alsS in the early development of Staphylococcus aureus UAMS-1 biofilm formation and the structural role of extracellular DNA and carbohydrates.

Extracellular DNA (eDNA) is an important component of the extracellular polymeric substance matrix and is important in the establishment and persistence of Staphylococcus aureus UAMS-1 biofilms. The aim of the study was to determine the temporal expression of genes involved in early biofilm formation and eDNA production. We used qPCR to investigate expression of agrB, which is associated with secreted virulence factors and biofilm dispersal, cidA, which is associated with biofilm adherence and genomic DNA release, and alsS, which is associated with cell lysis, eDNA release and acid tolerance. The contribution of eDNA to the stability of the biofilm matrix was assessed by digesting with DNase I (Pulmozyme) and quantifying structure by confocal microscopy and comstat image analysis. AgrB expression initially increased at 24 h but then dramatically decreased at 72 h in an inverse relationship to biomass, supporting its role in regulating biofilm dispersal. cidA and alsS expression steadily increased over 72 h, suggesting that eDNA was an important component of early biofilm development. DNase I had no effect on biomass, but did cause the biofilms to become more heterogeneous. Carbohydrates in the matrix appeared to play an important role in structural stability.

Reducing Staphylococcus aureus biofilm formation on stainless steel 316L using functionalized self-assembled monolayers.

Stainless steel 316L (SS316L) is a common material used in orthopedic implants. Bacterial colonization of the surface and subsequent biofilm development can lead to refractory infection of the implant. Since the greatest risk of infection occurs perioperatively, strategies that reduce bacterial adhesion during this time are important. As a strategy to limit bacterial adhesion and biofilm formation on SS316L, self-assembled monolayers (SAMs) were used to modify the SS316L surface. SAMs with long alkyl chains terminated with hydrophobic (-CH3) or hydrophilic (oligoethylene glycol) tail groups were used to form coatings and in an orthogonal approach, SAMs were used to immobilize gentamicin or vancomycin on SS316L for the first time to form an "active" antimicrobial coating to inhibit early biofilm development. Modified SS316L surfaces were characterized using surface infrared spectroscopy, contact angles, MALDI-TOF mass spectrometry and atomic force microscopy. The ability of SAM-modified SS316L to retard biofilm development by Staphylococcus aureus was functionally tested using confocal scanning laser microscopy with COMSTAT image analysis, scanning electron microscopy and colony forming unit analysis. Neither hydrophobic nor hydrophilic SAMs reduced biofilm development. However, gentamicin-linked and vancomycin-linked SAMs significantly reduced S. aureus biofilm formation for up to 24 and 48 h, respectively.

In vivo capsular switch in Streptococcus pneumoniae--analysis by whole genome sequencing.

Two multidrug resistant strains of Streptococcus pneumoniae - SV35-T23 (capsular type 23F) and SV36-T3 (capsular type 3) were recovered from the nasopharynx of two adult patients during an outbreak of pneumococcal disease in a New York hospital in 1996. Both strains belonged to the pandemic lineage PMEN1 but they differed strikingly in virulence when tested in the mouse model of IP infection: as few as 1000 CFU of SV36 killed all mice within 24 hours after inoculation while SV35-T23 was avirulent.Whole genome sequencing (WGS) of the two isolates was performed (i) to test if these two isolates belonging to the same clonal type and recovered from an identical epidemiological scenario only differed in their capsular genes? and (ii) to test if the vast difference in virulence between the strains was mostly - or exclusively - due to the type III capsule. WGS demonstrated extensive differences between the two isolates including over 2500 single nucleotide polymorphisms in core genes and also differences in 36 genetic determinants: 25 of which were unique to SV35-T23 and 11 unique to strain SV36-T3. Nineteen of these differences were capsular genes and 9 bacteriocin genes.Using genetic transformation in the laboratory, the capsular region of SV35-T23 was replaced by the type 3 capsular genes from SV36-T3 to generate the recombinant SV35-T3* which was as virulent as the parental strain SV36-T3* in the murine model and the type 3 capsule was the major virulence factor in the chinchilla model as well. On the other hand, a careful comparison of strains SV36-T3 and the laboratory constructed SV35-T3* in the chinchilla model suggested that some additional determinants present in SV36 but not in the laboratory recombinant may also contribute to the progression of middle ear disease. The nature of this determinants remains to be identified.

Differences in genotype and virulence among four multidrug-resistant Streptococcus pneumoniae isolates belonging to the PMEN1 clone.

We report on the comparative genomics and characterization of the virulence phenotypes of four S. pneumoniae strains that belong to the multidrug resistant clone PMEN1 (Spain(23F) ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinants.